Laboratory work physiological properties of the cell membrane. Comparison of membrane permeability of living and dead cells. Literature for students

1. Cell membranes, their types. Properties of membranes. Functions of membranes.

Morphological and physiological studies have shown that the cell membrane plays an important role in the functioning of the cell.

Membrane structures: nucleus, Golgi complex, ER, etc.

Membrane is a thin structure with a thickness of 7 nm. In terms of its chemical composition, the membrane contains 25% proteins, 25% phospholipids, 13% cholesterol, 4% lipids, 3% carbohydrates.

Structurally The membrane is based on a double layer of phospholipids. A feature of phospholipid molecules is that they have hydrophilic and hydrophobic parts. The hydrophilic parts contain polar groups (phosphate groups in phospholipids and hydroxide groups in cholesterols). Hydrophilic parts directed towards the surface. A hydrophobic (fat tails) are directed towards the center of the membrane.

The molecule has two fatty tails, and these hydrocarbon chains can be found in two configurations. Elongated - trans configuration(cylinder 0.48 nm). The second type is the gauche-trans-gauche configuration. In this case, the two fat tails diverge and the area increases to 0.58 nm.

Under normal conditions, lipid molecules have a liquid crystalline form. And in this state they have mobility. Moreover, they can both move within their layer and turn over. As the temperature decreases, the membrane transitions from a liquid state to a jelly-like state, and this reduces the mobility of the molecule.

When a lipid molecule moves, microstrips are formed, which are called kings, into which substances can be captured. The lipid layer in the membrane is a barrier to water-soluble substances, but allows lipid-soluble substances to pass through..

In addition to lipids, the membrane also contains protein molecules. These are mainly glycoproteins.

Integral proteins pass through both layers. Other proteins are partially immersed in either the outer or inner layer. They are called peripheral proteins.

This membrane model is called liquid crystal model. Functionally, protein molecules perform structural, transport, and enzymatic functions. In addition, they form ion channels ranging from 0.35 to 0.8 nm in diameter through which ions can pass. Channels have their own specialization. Integral proteins are involved in active transport and facilitated diffusion.

Peripheral proteins on the inner side of the membrane are characterized by enzymatic function. On the inside there are antigenic (antibodies) and receptor functions.

Carbon chains can attach to protein molecules, and then they are formed glycoproteins. Or to lipids, then they are called glycolipids.

Main functions cell membranes will be:

1. Barrier function

2. Passive and active transfer of substances.

3. Metabolic function (due to the presence of enzyme systems in them)

4. Membranes are involved in the creation of electrical potentials at rest, and when excited, action currents.

5. Receptor function.

6. Immunological (associated with the presence of antigens and the production of antibodies).

7. Provide intercellular interaction and contact inhibition.

When homogeneous cells come into contact, cell division is inhibited. This function is lost in cancer cells. In addition, cancer cells come into contact not only with their own cells, but also with other cells, infecting them.

Function of membrane permeability. Transport.

Transport of substances across membranes can be passive or active.

Passive transfer substances pass through membranes without energy consumption in the presence of gradients (differences in the concentrations of substances, differences in the electrochemical gradient, in the presence of a pressure gradient and osmotic gradient). In this case, passive transport is carried out using:

Diffusion.

Filtration. It is carried out in the presence of a difference in hydrostatic pressure.

Osmosis. During osmosis, the solvent moves. That is, water from a pure solution will move into a solution with a higher concentration.

In all these cases no energy consumption occurs. Substances pass through the pores in the membrane.

There are pores with slow conductivity in the membrane, but there are not many such pores in the membrane. Most channels in the membrane also have a gate mechanism in their structure that closes the channel. These channels can be controlled in two ways: respond to changes in charge (electrically excitable or voltage-gated channels). In another case, the gate in the channel opens when a chemical (chemoexcitable or ligand-gated) is attached.

Active transfer substances across the membrane is associated with the transport of substances against a gradient.

For active transport, integral proteins that have enzymatic functions are used. ATP is used as energy. Integral proteins have special mechanisms (proteins) that are activated either when the concentration of a substance increases outside the cell, or when it decreases inside.

Quiescent currents.

Membrane potential. The membrane is charged positively on the outside and negatively charged on the inside. 70-80 mV.

Fault current is the charge difference between the undamaged and damaged. The damaged one is negatively charged, relative to the intact one.

Metabolic current is the difference in potentials due to the unequal intensity of metabolic processes.

The origin of the membrane potential is explained in terms of membrane-ion theory, which takes into account the unequal permeability of the membrane for ions and the different composition of ions in the intracellular and intercellular fluid. It has been established that both intracellular and intercellular fluid have the same number of positive and negative ions, but the composition is different. External liquid: Na + , Cl - Internal liquid: K + , A - (organic anions)

At rest, the membrane is differently permeable to ions. Potassium has the greatest permeability, followed by sodium and chlorine. Membranes are not permeable to organic anions.

Due to increased permeability to potassium ions, they leave the cell. As a result, organic matter accumulates inside. anions. The result is a potential difference (potassium diffusion potential) that continues as long as it can escape.

The calculated potassium potential is -90 mV. And the practical potential is -70 mV. This suggests that another ion is also involved in creating the potential.

In order to restrain the potential in the membrane, the cell must work, because the movement of potassium ions from the cell, and sodium ions into the cell, would lead to a violation of the equality of sign. The membranes are polarized. The charge on the outside will be positive and the charge on the outside will be negative.

The state of the electrical charge of the membrane.

Reversal or overshoot - changing the sign of the charge. Return to the original charge - repolarization.

Excitation currents.

When a stimulus acts on the membrane, short-term excitation occurs. The excitation process is local and spreads along the membrane, and then depolarizes. As the excitation moves, a new section of the membrane is depolarized, etc. The action current is a two-phase current.

In each phase of the action current, a local response can be distinguished, which is replaced by a peak potential, and the peak potential is followed by a negative and positive trace potential. Occurs when exposed to a stimulus. To explain the action current, it was proposed membrane-mon theory(Hodgey, Huxley, Katz). They showed that action potential is greater than resting potential. When a stimulus acts on the membrane, the charge shifts to the membrane (partial depolarization) and this causes the opening of sodium channels. Sodium penetrates into the cell, gradually reducing the charge on the membrane, but the action potential does not arise with any action, but only with a critical value (change by 20-30 mV) - critical depolarization. In this case, almost all sodium channels open and in this case sodium begins to penetrate into the cell like an avalanche. Complete depolarization occurs. The process does not stop here, but continues to enter the cell and charges up to +40. At the top of the peak potential, the h gate closes. At this potential value, the potassium gate opens in the membrane. And since Ka + is larger inside, Ka + begins to leave the cell, and the charge will begin to return to its original value. It goes fast at first and then slows down. This phenomenon is called negative tail potential. Then the charge is restored to its original value, and after this a positive trace potential is recorded, characterized by increased permeability to potassium. A state of membrane hyperpolarization occurs (positive trace potential). The movement of ions occurs passively. During one excitation, 20,000 sodium ions enter the cell, and 20,000 potassium ions leave the cell.

The pumping mechanism is necessary to restore concentration. 3 positive sodium ions are brought in and 2 potassium ions come out during active transport.

The excitability of the membrane changes, and therefore the action potential. During the local response, a gradual increase in excitation occurs. During the peak response, the excitation disappears.

With a negative trace potential, excitability will increase again, because the membrane is again partially depolarized. In the phase of positive light potential, a decrease in excitability occurs. Under these conditions, excitability decreases.

Speed ​​of the excitatory process - lability. A measure of lability - the number of excitations per unit time. Nerve fibers reproduce from 500 to 1000 impulses per second. Different tissues have different lability.

2. Receptors, their classification: by localization (membrane, nuclear), by the mechanism of development of processes (iono- and metabotropic), by the speed of signal reception (fast, slow), by the type of receptor substances.

The cell's receipt of a signal from primary messengers is ensured by special receptor proteins, for which primary messengers are ligands. To ensure receptor function, protein molecules must meet a number of requirements:

• have high selectivity for the ligand;
• the kinetics of ligand binding should be described by a saturation curve corresponding to the state of full occupancy of all receptor molecules, the number of which is limited on the membrane;
• receptors must have tissue specificity, reflecting the presence or absence of these functions in the cells of the target organ;
• The binding of the ligand and its cellular (physiological) effect must be reversible, and the affinity parameters must correspond to the physiological concentrations of the ligand.

Cellular receptors are divided into the following classes:

• membrane
• receptor tyrosine kinases
• G protein-coupled receptors
• ion channels
• cytoplasmic
• nuclear

Membrane receptors recognize large (for example, insulin) or hydrophilic (for example, adrenaline) signaling molecules that cannot independently penetrate the cell. Small hydrophobic signaling molecules (for example, triiodothyronine, steroid hormones, CO, NO) are able to enter the cell due to diffusion. The receptors for such hormones are usually soluble cytoplasmic or nuclear proteins. After the ligand binds to the receptor, information about this event is transmitted further along the chain and leads to the formation of a primary and secondary cellular response.

The two main classes of membrane receptors are metabotropic receptors and ionotropic receptors.

Ionotropic receptors are membrane channels that open or close upon binding to a ligand. The resulting ionic currents cause changes in the transmembrane potential difference and, as a result, cell excitability, and also change intracellular ion concentrations, which can secondarily lead to the activation of intracellular mediator systems. One of the most fully studied ionotropic receptors is the n-cholinergic receptor.

Structure of a G protein consisting of three types of units (heterotrimeric) - αt/αi (blue), β (red) and γ (green)

Metabotropic receptors are associated with systems of intracellular messengers. Changes in their conformation upon binding to a ligand lead to the launch of a cascade of biochemical reactions and, ultimately, a change in the functional state of the cell. Main types of membrane receptors:

Heterotrimeric G protein-coupled receptors (eg, vasopressin receptor).

Receptors with intrinsic tyrosine kinase activity (for example, insulin receptor or epidermal growth factor receptor).

G protein-coupled receptors are transmembrane proteins having 7 transmembrane domains, an extracellular N terminus and an intracellular C terminus. The ligand binding site is located on the extracellular loops, the G protein binding domain is located near the C-terminus in the cytoplasm.

Activation of the receptor causes its α-subunit to dissociate from the βγ-subunit complex and thus become activated. After this, it either activates or, on the contrary, inactivates the enzyme that produces second messengers.

Receptors with tyrosine kinase activity phosphorylate subsequent intracellular proteins, often also protein kinases, and thus transmit a signal into the cell. Structurally, these are transmembrane proteins with one membrane domain. As a rule, homodimers, the subunits of which are linked by disulfide bridges.

3. Ionotropic receptors, metabotropic receptors and their varieties. Systems of secondary messengers of the action of metabotropic receptors (cAMP, c GMP, inositol-3-phosphate, diacylglycerol, Ca++ ions).

Receptors for neurotransmitters are located on the membranes of neurons or target cells (muscle or glandular cells). Their localization can be on both postsynaptic and presynaptic membranes. So-called autoreceptors are often located on presynaptic membranes, which regulate the release of the same transmitter from the presynaptic ending. But there are also heteroautoreceptors that also regulate the release of a mediator, but in these receptors the release of one mediator is regulated by another mediator or neuromodulator.

Most receptors are membrane-bound oligomeric proteins that bind a ligand (neurotransmitter) with high affinity and high selectivity. As a result of this interaction, a cascade of intracellular changes is triggered. Receptors are characterized by affinity for the ligand, number, saturability and ability to dissociate the receptor-ligand complex. Some receptors have isoforms that differ in their affinity for certain ligands. These isoforms can be found in the same tissue.

Ligands are substances that selectively interact with a given receptor. If a pharmacological substance activates a given receptor, it is an agonist for it, and if it reduces its activity, it is an antagonist.

Binding of a ligand to a receptor leads to a change in the conformation of the receptor, which either opens ion channels or triggers a cascade of reactions leading to changes in metabolism.

There are ionotropic and metabotropic receptors.

Ionotropic receptors. Due to the formation of the postsynaptic potential, the corresponding ion channel opens either immediately upon the action of the mediator, or through the activation of the G protein. In this case, the receptor either forms an ion channel itself or is associated with it. After the ligand attaches and the receptor is activated, the channel for the corresponding ion opens. As a result, a postsynaptic potential is formed on the membrane. Ionotropic receptors are a way of rapid signal transmission and formation of PSP without changing metabolic processes in the cell.

Metabotropic receptors. This is a more complex signal transmission pathway. In this case, after binding of the ligand to the receptor, the phosphorylation-dephosphorylation cascade is activated. This occurs either directly or through secondary messengers, for example, through tyrosine kinase, or through cAMP, or cGMP, or inositol triphosphate, or diacylglycerol, or through an increase in intracellular calcium, which ultimately leads to the activation of protein kinases. Phosphorylation most often involves the activation of cAMP-dependent or diacylglycerol-dependent protein kinases. These effects develop more slowly and last longer.

The affinity of the receptor for the corresponding neurotransmitter can change in the same way as for hormones, for example, due to allosteric changes in the receptor or other mechanisms. Therefore, receptors are now referred to as mobile and easily changeable structures. Being part of the membrane, receptor proteins can interact with other membrane proteins (the so-called internalization of receptors). Neuromodulators, like neurotransmitters, can influence the number and sensitivity of receptors. The prolonged presence of large amounts of a neurotransmitter or neuromodulator can reduce their sensitivity (down-regulation), and the lack of ligands can increase their sensitivity (up-regulation).

4. Ion channels, their structure. Classification of ion channels. Sodium and potassium channels.

Structure and functions of ion channels. Na + , K + , Ca 2+ , Cl - ions penetrate into the cell and exit through special liquid-filled channels. The size of the channels is quite small (diameter 0.5-0.7 nm). Calculations show that the total area of ​​the channels occupies an insignificant part of the surface of the cell membrane.

The function of ion channels is studied in various ways. The most common is the voltage clamp method, or “voltage-clamp” (Fig. 2.2). The essence of the method is that, with the help of special electronic systems, the membrane potential is changed and fixed at a certain level during the experiment. In this case, the magnitude of the ionic current flowing through the membrane is measured. If the potential difference is constant, then in accordance with Ohm's law, the current value is proportional to the conductivity of the ion channels. In response to stepwise depolarization, certain channels open and the corresponding ions enter the cell along an electrochemical gradient, i.e., an ion current arises that depolarizes the cell. This change is detected by a control amplifier and an electric current is passed through the membrane, equal in magnitude but opposite in direction to the membrane ion current. In this case, the transmembrane potential difference does not change. The combined use of voltage clamp and specific ion channel blockers led to the discovery of various types of ion channels in the cell membrane.

Currently, many types of channels for various ions have been installed (Table 2.1). Some of them are very specific, while others, in addition to the main ion, can allow other ions to pass through.

Studying the function of individual channels is possible using the method of local fixation of the “path-clamp” potential; rice. 2.3, A). A glass microelectrode (micropipette) is filled with saline solution, pressed against the surface of the membrane and a slight vacuum is created. In this case, part of the membrane is sucked to the microelectrode. If there is an ion channel in the suction zone, then the activity of a single channel is recorded. The system of stimulation and recording of channel activity differs little from the system of voltage recording.

Table 2.1. The most important ion channels and ion currents of excitable cells

Channel type	Function		Channel blocker
Potassium (at rest)	Generation of resting potential	IK+ (leakage)
Sodium	Action potential generation
Calcium	Generation of slow potentials		D-600, verapamil
Potassium (delayed straightening)	Ensuring repolarization	IK+ (delay)
Potassium calcium-activated	Limitation of depolarization caused by Ca 2+ current

Note. TEA - tetraethylammonium; TTX - tetrodotoxin.

The outer part of the canal is relatively accessible for study; studying the inner part presents significant difficulties. P. G. Kostyuk developed a method of intracellular dialysis, which makes it possible to study the function of the input and output structures of ion channels without the use of microelectrodes. It turned out that the part of the ion channel open to the extracellular space differs in its functional properties from the part of the channel facing the intracellular environment.

It is ion channels that provide two important properties of the membrane: selectivity and conductivity.

Selectivity or selectivity, channel is provided by its special protein structure. Most channels are electrically controlled, that is, their ability to conduct ions depends on the magnitude of the membrane potential. The channel is heterogeneous in its functional characteristics, especially with regard to the protein structures located at the entrance to the channel and at its exit (the so-called gate mechanisms).

5. The concept of excitability. Parameters of excitability of the neuromuscular system: threshold of irritation (rheobase), useful time (chronaxy). Dependence of the strength of irritation on the time of its action (Goorweg-Weiss curve). Refractoriness.

Excitability- the ability of a cell to respond to irritation by forming an action potential and a specific reaction.

1) local response phase - partial depolarization of the membrane (entry of Na + into the cell). If you apply a small stimulus, the response is stronger.

Local depolarization is the exaltation phase.

2) phase of absolute refractoriness - the property of excitable tissues not to form AP under any stimulus strength

3) phase of relative refractoriness.

4) slow repolarization phase - irritation - again a strong response

5) hyperpolarization phase - excitability is less (subnormal), the stimulus should be large.

Functional lability- assessment of tissue excitability through the maximum possible number of PD per unit time.

Excitation laws:

1) the law of force - the strength of the stimulus must be threshold or suprathreshold (the minimum amount of force that causes excitation). The stronger the stimulus, the stronger the excitation - only for tissue associations (nerve trunk, muscle, exception - SMC).

2) the law of time - the duration of the current stimulus must be sufficient for the occurrence of excitation.

There is an inversely proportional relationship between force and time within the boundaries between minimum time and minimum force. The minimum force is rheobase - a force that causes excitation and does not depend on duration. The minimum time is useful time. Chronaxy is the excitability of a particular tissue; the time at which excitation occurs is equal to two rheobases.

The greater the force, the greater the response up to a certain value.

Factors creating MSP:

1) difference in concentrations of sodium and potassium

2) different permeability for sodium and potassium

3) operation of the Na-K pump (3 Na + is removed, 2 K + is returned).

The relationship between the strength of the stimulus and the duration of its impact, necessary for the occurrence of a minimal response of a living structure, can be very clearly traced on the so-called force-time curve (Goorweg-Weiss-Lapik curve).

From the analysis of the curve it follows that, no matter how great the strength of the stimulus, if the duration of its influence is insufficient, there will be no response (the point to the left of the ascending branch of the hyperbola). A similar phenomenon is observed with prolonged exposure to subthreshold stimuli. The minimum current (or voltage) capable of causing excitation is called rheobase by Lapik (ordinate segment OA). The shortest period of time during which a current equal in strength to twice the rheobase causes excitation in the tissue is called chronaxy (abscissa segment OF), which is an indicator of the threshold duration of irritation. Chronaxy is measured in δ (thousandths of a second). The magnitude of the chronaxy can be used to judge the rate at which excitation occurs in the tissue: the smaller the chronaxy, the faster the excitation occurs. The chronaxy of human nerve and muscle fibers is equal to thousandths and ten-thousandths of a second, and the chronaxy of so-called slow tissues, for example, the muscle fibers of a frog's stomach, is hundredths of a second.

The determination of the chronaxy of excitable tissues has become widespread not only in experiment, but also in sports physiology and in the clinic. In particular, by measuring muscle chronaxy, a neurologist can determine the presence of motor nerve damage. It should be noted that the stimulus can be quite strong, have a threshold duration, but a low rate of increase in time to the threshold value; in this case, excitation does not occur. The adaptation of excitable tissue to a slowly increasing stimulus is called accommodation. Accommodation is due to the fact that during the increase in the strength of the stimulus, active changes have time to develop in the tissue, increasing the threshold of irritation and preventing the development of excitation. Thus, the rate of increase in stimulation over time, or the gradient of stimulation, is essential for the occurrence of excitation.

Law of irritation gradient. The reaction of a living formation to a stimulus depends on the gradient of stimulation, i.e., on the urgency or steepness of the increase in the stimulus over time: the higher the gradient of stimulation, the stronger (up to certain limits) the response of the excitable formation.

Consequently, the laws of stimulation reflect the complex relationship between the stimulus and the excitable structure during their interaction. For excitation to occur, the stimulus must have a threshold strength, have a threshold duration, and have a certain rate of increase over time.

6. Ion pumps (ATPases):K+- Na+-evaya,Ca2+-eva (plasmolemma and sarcoplasmic reticulum),H+- K+-exchanger.

According to modern concepts, biological membranes contain ion pumps that operate using the free energy of ATP hydrolysis - special systems of integral proteins (transport ATPases).

Currently, three types of electrogenic ion pumps are known that actively transport ions through the membrane (Fig. 13).

The transfer of ions by transport ATPases occurs due to the coupling of transfer processes with chemical reactions, due to the energy of cell metabolism.

When K+-Na+-ATPase operates, two potassium ions are transferred into the cell due to the energy released during the hydrolysis of each ATP molecule and three sodium ions are simultaneously pumped out of the cell. This creates an increased concentration of potassium ions in the cell compared to the intercellular environment and a decreased concentration of sodium, which is of great physiological importance.

Signs of a “biopump”:

1. Movement against the electrochemical potential gradient.

2. the flow of matter is associated with the hydrolysis of ATP (or other energy source).

3. asymmetry of the transport vehicle.

4. The pump in vitro is capable of hydrolyzing ATP only in the presence of those ions that it transports in vivo.

5. When the pump is embedded in an artificial environment, it is able to maintain selectivity.

The molecular mechanism of operation of ion ATPases is not fully understood. Nevertheless, the main stages of this complex enzymatic process can be traced. In the case of K+-Na+-ATPase, there are seven stages of ion transfer associated with ATP hydrolysis.

The diagram shows that the key stages of the enzyme are:

1) formation of an enzyme complex with ATP on the inner surface of the membrane (this reaction is activated by magnesium ions);

2) binding of three sodium ions by the complex;

3) phosphorylation of the enzyme with the formation of adenosine diphosphate;

4) revolution (flip-flop) of the enzyme inside the membrane;

5) the reaction of ion exchange of sodium to potassium, occurring on the outer surface of the membrane;

6) reverse revolution of the enzyme complex with the transfer of potassium ions into the cell;

7) return of the enzyme to its original state with the release of potassium ions and inorganic phosphate (P).

Thus, during a complete cycle, three sodium ions are released from the cell, the cytoplasm is enriched with two potassium ions, and hydrolysis of one ATP molecule occurs.

7. Membrane potential, magnitude and origin.

Many theories have been proposed to explain the origin of biopotentials. The membrane theory proposed by the German researcher Bernstein (1902, 1912) was most fully experimentally substantiated. In the modern period, this theory has been modified and experimentally developed by Hodgkin, Huxley, Katz (1949-1952).

It has been established that the basis of bioelectric phenomena is the uneven distribution (asymmetry) of ions in the cytoplasm of the cell and its environment. Thus, the protoplasm of nerve and muscle cells contains 30-50 times more potassium ions, 8-10 times less sodium ions and 50 times less chlorine ions than extracellular fluid. In addition, the cell cytoplasm contains organic anions (large molecular compounds carrying a negative charge), which are absent in the extracellular environment.

Proponents of the membrane theory believe that the main reason for ion asymmetry is the presence of a cell membrane with specific properties.

The cell membrane is a compacted layer of cytoplasm, the thickness of which is about 10 nm (100 A). The use of electron microscopic research methods made it possible to determine the fine structure of the membrane (Fig. 55). The cell membrane consists of a double layer of phospholipid molecules, which is covered on the inside with a layer of protein molecules, and on the outside with a layer of complex carbohydrate molecules - mucopolysaccharides. The membrane has special channels - “pores”, through which water and ions penetrate into the cell. It is assumed that there are special channels for each ion. In this regard, the permeability of the membrane for certain ions will depend on the size of the pores and the diameters of the ions themselves.

In a state of relative physiological rest, the membrane has increased permeability to potassium ions, while its permeability to sodium ions is sharply reduced.

Thus, the characteristics of the permeability of the cell membrane, as well as the size of the ions themselves, are one of the reasons that ensure the asymmetry of the distribution of ions on both sides of the cell membrane. Ionic asymmetry is one of the main causes of the resting potential, with the leading role played by the uneven distribution of potassium ions.

Hodgkin performed classical experiments on giant squid nerve fiber. The concentration of potassium ions inside the fiber and in the surrounding liquid was equalized - the resting potential disappeared. If the fiber was filled with an artificial saline solution, similar in composition to intracellular fluid, a potential difference was established between the inner and outer sides of the membrane, approximately equal to the resting potential of a normal fiber (50-80 mV).

The mechanism by which an action potential occurs is much more complex. The main role in the occurrence of action currents belongs to sodium ions. When exposed to a stimulus of threshold strength, the permeability of the cell membrane for sodium ions increases 500 times and exceeds the permeability for potassium ions by 10-20 times. In this regard, sodium rushes into the cell like an avalanche, which leads to a recharge of the cell membrane. The outer surface is charged negatively relative to the inner one. Depolarization of the cell membrane occurs, accompanied by a reversal of the membrane potential. Membrane potential reversal refers to the number of millivolts (mV) by which the action potential exceeds the resting potential. Restoration of the initial level of membrane potential (repolarization) is carried out due to a sharp decrease in sodium permeability (inactivation) and the active transfer of sodium ions from the cell cytoplasm to the environment.

Evidence for the sodium action potential hypothesis was also obtained by Hodgkin. Indeed, if the action potential is sodium in nature, then by varying the concentration of sodium ions, the magnitude of the action potential can be changed. It turned out that when replacing 2/3 of sea water, which is the normal environment for the giant squid axon, with an isotonic dextrose solution, i.e., when the sodium concentration in the environment changes by 2/3, the action potential is reduced by half.

Thus, the emergence of biopotentials is a function of a biological membrane that has selective permeability. The magnitude of the resting potential and action potential is determined by ionic asymmetry in the cell-environment system.

8. Electrical phenomena in nervous and muscle tissues during excitement. Action potential, its magnitude, phases and duration. The relationship between the phases of the action potential and the phases of excitability.

We have already shown above that excitation in nerve and muscle fibers is carried out using electrical impulses propagating along the surface membrane. The transfer of excitation from nerve to muscle is based on a different mechanism. It is carried out as a result of the release by nerve endings of highly active chemical compounds - mediators of the nerve impulse. At skeletal muscle synapses, such a transmitter is acetylcholine (ACh).

There are three main structural elements in the neuromuscular synapse - presynaptic membrane on the nerve postsynaptic membrane on the muscle, between them - synaptic cleft . The shape of the synapse can be varied. At rest, ACh is contained in so-called synaptic vesicles inside the end plate of the nerve fiber. The cytoplasm of the fiber with synaptic vesicles floating in it is separated from the synaptic cleft by a presynaptic membrane. When the presynaptic membrane is depolarized, its charge and permeability changes, the vesicles come close to the membrane and pour into the synaptic cleft, the width of which reaches 200-1000 angstroms. The transmitter begins to diffuse through the gap to the postsynaptic membrane.

The postsynaptic membrane is not electrogenic, but is highly sensitive to the transmitter due to the presence of so-called cholinergic receptors - biochemical groups that can selectively react with ACh. The latter reaches the postsynaptic membrane in 0.2-0.5 ms. (so-called "synaptic delay") and, interacting with cholinergic receptors, causes a change in the permeability of the membrane for Na, which leads to depolarization of the postsynaptic membrane and the generation of a depolarization wave on it, which is called excitatory postsynaptic potential, (EPSP), the value of which exceeds the Ec of neighboring, electrogenic areas of the muscle fiber membrane. As a result, an action potential (AP) arises in them, which spreads over the entire surface of the muscle fiber, then causing its contraction, initiating the so-called process. electromechanical coupling (Capling). The transmitter in the synaptic cleft and on the postsynaptic membrane works for a very short time, as it is destroyed by the enzyme cholinesterase, which prepares the synapse to receive a new portion of the transmitter. It has also been shown that part of the unreacted ACh can return to the nerve fiber.

With very frequent rhythms of stimulation, postsynaptic potentials can be summed up, since cholinesterase does not have time to completely break down the ACh released in the nerve endings. As a result of this summation, the postsynaptic membrane becomes more and more depolarized. In this case, the neighboring electrogenic areas of the muscle fiber enter a state of depression similar to that which develops during prolonged action of a direct current cathode (Verigo cathodic depression).

Excitation in a tissue is manifested in the appearance of a function specific to it (conduction of excitation by nervous tissue, muscle contraction, gland secretion) and nonspecific reactions (generation of an action potential, metabolic changes).

Action current (PD and ECP) ​​is an electrical current that occurs in nerve, muscle and some plant cells between their excited and neighboring resting areas. Caused by changes in membrane ionic permeability and potential that develop in the excited area. Plays an important role in the propagation of action potential along the cell (fiber). An action potential is a shift in membrane potential that occurs in tissue under the action of a threshold and suprathreshold stimulus, which is accompanied by recharging of the cell membrane.

When exposed to a threshold or suprathreshold stimulus, the permeability of the cell membrane for ions changes to varying degrees. For Na ions it increases by 400-500 times, and the gradient increases quickly, for K ions - by 10-15 times, and the gradient develops slowly. As a result, Na ions move into the cell, K ions move out of the cell, which leads to recharging of the cell membrane. The outer surface of the membrane carries a negative charge, while the inner surface carries a positive charge. Accurate measurements showed that the amplitude of the action potential is 30-50 mV higher than the resting potential.

PD phases. PD consists of 2 phases:

1. Depolarization phase. Corresponds to a rapid change in membrane potential (membrane depolarization) of approximately 110 mV. The membrane potential changes from a resting level (about -70 mV) to a value close to the equilibrium potential - the potential at which the incoming current takes on a zero value (ENa + (about 40 mV)).

2. Repolarization phase. The membrane potential again reaches the resting level (the membrane is repolarized), after which hyperpolarization occurs to a value approximately 10 mV less (more negative) than the resting potential, i.e. approximately -80 mV.

The duration of the action potential in nerve and skeletal muscle fibers varies from 0.1 to 5 ms, while the repolarization phase is always longer than the depolarization phase.

The relationship between the phases of action potential and excitability. The level of cell excitability depends on the AP phase. During the local response phase, excitability increases. This phase of excitability is called latent addition. During the AP repolarization phase, when all sodium channels open and sodium ions rush into the cell like an avalanche, no stimulus, even a very strong one, can stimulate this process. Therefore, the phase of depolarization corresponds to the phase of absolute refractoriness. During the repolarization phase, an increasing part of the sodium channels closes. However, they can reopen under the influence of a suprathreshold stimulus. This corresponds to the phase of relative refractoriness. During trace depolarization, the MP is at a critical level, so even subthreshold stimuli can cause cell excitation. Consequently, at this moment her excitability is increased. This phase is called the supernormal excitability phase. At the moment of trace hyperpolarization, the MP is higher than the initial level. She is in a phase of subnormal excitability.

9. The structure of skeletal muscles and their innervation. Motor unit. Physiological properties of muscles, their characteristics in a newborn.

Morpho-functional classification of muscles:

1. Cross-striped

a) skeletal - multinucleated cells, cross-striated, the nuclei are closer to the sarcolemma. Weight 40%.

b) cardiac - cross-striated, mononuclear cells, the nucleus in the center. Weight 0.5%.

2. Smooth - mononuclear cells, do not have transverse striations. They are part of other organs. Total weight 5-10%.

General properties of muscles.

1) Excitability. PP = - 90mV. AP amplitude = 120 mV - sign reversal +30 mV.

2) Conductivity - the ability to conduct PD across the cell membrane (3-5 m/s). Provides delivery of PD to T-tubules and from them to L-tubules that release calcium.

3) Contractility - the ability to shorten or develop tension when excited.

4) Elasticity - the ability to return to its original length.

Functions of skeletal muscles:

1. Movement of the body in space

2. Moving body parts relative to each other

3. Maintaining the posture

4. Heat generation

5. Movement of blood and lymph (dynamic work)

6. Participation in ventilation

7. Protection of internal organs

8. Anti-stress factor

Levels of skeletal muscle organization:

The whole muscle is surrounded by the epimysium and is approached by blood vessels and nerves. Individual muscle bundles are covered with perimysium. A bundle of cells (muscle fiber or symplast) - covered with endomysium. The cell contains myofibrils from myofilaments, the main proteins - actin, myosin, tropomyosin, troponin, calcium ATPase, creatine phosphokinase, structural proteins.

In a muscle, motor units (motor, neuromotor units) are distinguished - this is a functional association of a motor neuron, its axon and muscle fibers innervated by this axon. These muscle fibers can be located in different parts (bundles) of the muscle.

A motor unit (MU) is the functional unit of skeletal muscle. ME includes a motor neuron and the group of muscle fibers innervated by it.

Types of muscle fibers:

1) slow phasic fibers of the oxidative type

2) fast phasic fibers of the oxidative type (type 2a)

3) fast phasic fibers of the glycolytic type (type 2b)

4) tonic fibers

Mechanisms of muscle contraction.

A) single muscle fiber

B) whole muscle

Skeletal muscle has the following essential properties:

1) excitability - the ability to respond to a stimulus by changing ionic conductivity and membrane potential. Under natural conditions, this irritant is the neurotransmitter acetylcholine.

2) conductivity - the ability to conduct the action potential along and deep into the muscle fiber along the T-system;

3) contractility - the ability to shorten or develop tension when excited;

4) elasticity - the ability to develop tension when stretched.

10. Modes of muscle contraction: isotonic and isometric. Absolute muscle strength. Age-related changes in muscle strength.

The contractility of a skeletal muscle is characterized by the force of contraction that the muscle develops (usually assessed overall strength which a muscle can develop, and absolute, i.e., the force per 1 cm 2 of the cross section). length of shortening, degree of tension of the muscle fiber, rate of shortening and development of tension, rate of relaxation. Since these parameters are largely determined by the initial length of muscle fibers and the load on the muscle, studies of muscle contractility are carried out in various modes.

Irritation of a muscle fiber by a single threshold or supra-threshold stimulus leads to the occurrence of a single contraction, which consists of several periods (Fig. 2.23). The first, the latent period, is the sum of time delays caused by the excitation of the muscle fiber membrane, the propagation of PD through the T-system into the fiber, the formation of inositol triphosphate, an increase in the concentration of intracellular calcium and activation of cross bridges. For the frog sartorius muscle, the latency period is about 2 ms.

The second is the period of shortening, or development of tension. In the case of free shortening of the muscle fiber we speak of isotonic contraction mode, in which the tension practically does not change, and only the length of the muscle fiber changes. If the muscle fiber is fixed on both sides and cannot be freely shortened, then it is said to be isometric contraction mode Strictly speaking, with this mode of contraction, the length of the muscle fiber does not change, while the size of the sarcomeres changes due to the sliding of actin and myosin filaments relative to each other. In this case, the resulting tension is transferred to elastic elements located inside the fiber. Cross-bridges of myosin filaments, actin filaments, Z-plates, longitudinally located sarcoplasmic reticulum and the sarcolemma of muscle fibers have elastic properties.

In experiments on an isolated muscle, stretching of the connective tissue elements of the muscle and tendons is revealed, to which the tension developed by the transverse bridges is transmitted.

In the human body, isotonic or isometric contraction does not occur in isolated form. As a rule, the development of tension is accompanied by a shortening of muscle length - auxotonic mode contraction

The third is a period of relaxation, when the concentration of Ca 2+ ions decreases and the myosin heads are disconnected from the actin filaments.

It is believed that for a single muscle fiber the tension developed by any sarcomere is equal to the tension in any other sarcomere. Since the sarcomeres are connected in series, the rate at which a muscle fiber contracts is proportional to the number of its sarcomeres. Thus, during a single contraction, the rate of shortening of a long muscle fiber is higher than that of a shorter one. The amount of force developed by a muscle fiber is proportional to the number of myofibrils in the fiber. During muscle training, the number of myofibrils increases, which is the morphological substrate for increasing the force of muscle contraction. At the same time, the number of mitochondria increases, increasing the endurance of the muscle fiber during physical activity.

In an isolated muscle, the magnitude and speed of a single contraction are determined by a number of additional factors. The magnitude of a single contraction will primarily be determined by the number of motor units involved in the contraction. Since muscles consist of muscle fibers with different levels of excitability, there is a certain relationship between the magnitude of the stimulus and the response. An increase in contraction force is possible up to a certain limit, after which the contraction amplitude remains unchanged as the stimulus amplitude increases. In this case, all muscle fibers that make up the muscle take part in contraction.

The importance of the participation of all muscle fibers in contraction is shown when studying the dependence of the speed of shortening on the magnitude of the load.

When a second stimulus is applied during the period of shortening or development of muscle tension, the summation of two successive contractions occurs and the resulting response in amplitude becomes significantly higher than with a single stimulus; If a muscle fiber or muscle is stimulated with such a frequency that repeated stimuli will occur during the period of shortening or development of tension, then a complete summation of single contractions occurs and develops smooth tetanus (Fig. 2.25, B). Tetanus is a strong and prolonged muscle contraction. It is believed that this phenomenon is based on an increase in the concentration of calcium inside the cell, which allows the interaction between actin and myosin and the generation of muscle force by cross bridges to take place for a sufficiently long time. When the frequency of stimulation is reduced, it is possible that a repeated stimulus is applied during a period of relaxation. In this case, summation of muscle contractions will also occur, but a characteristic retraction on the muscle contraction curve will be observed (Fig. 2.25, D) - incomplete summation, or serrated tetanus.

With tetanus, summation of muscle contractions occurs, while the action potential of muscle fibers is not summed up.

Under natural conditions, single contractions of skeletal muscles do not occur. Addition occurs, or superposition, abbreviations of individual neuromotor units. In this case, the force of contraction can increase both due to a change in the number of motor units involved in the contraction, and due to a change in the frequency of impulses of motor neurons. If the impulse frequency increases, a summation of contractions of individual motor units will be observed.

One of the reasons for the increase in contraction force in natural conditions is the frequency of impulses generated by motor neurons. The second reason for this is an increase in the number of excited motor neurons and synchronization of the frequency of their excitation. An increase in the number of motor neurons corresponds to an increase in the number of motor units involved in contraction, and an increase in the degree of synchronization of their excitation contributes to an increase in the amplitude during the superposition of the maximum contraction developed by each motor unit separately.

The force of contraction of an isolated skeletal muscle, other things being equal, depends on the initial length of the muscle. Moderate stretching of a muscle leads to the fact that the force it develops increases compared to the force developed by an unstretched muscle. There is a summation of passive tension, caused by the presence of elastic components of the muscle, and active contraction. The maximum contractile force is achieved when the sarcomere size is 2-2.2 µm (Fig. 2.26). An increase in sarcomere length leads to a decrease in the force of contraction, since the area of ​​mutual overlap of actin and myosin filaments decreases. With a sarcomere length of 2.9 µm, the muscle can develop a force equal to only 50% of the maximum possible.

Under natural conditions, the force of contraction of skeletal muscles when stretched, for example during massage, increases due to the work of gamma efferents.

Absolute muscle strength is the ratio of the maximum muscle strength to its physiological diameter, i.e. the maximum load that a muscle can lift divided by the total area of ​​all muscle fibers. The force of contraction does not remain constant throughout life. As a result of prolonged activity, the performance of skeletal muscles decreases. This phenomenon is called fatigue. At the same time, the force of contraction decreases, the latent period of contraction and the period of relaxation increase.

11. Single muscle contractions, its phases. Phases of changes in muscle excitability. Features of a single contraction in newborns.

Stimulation of a muscle or the motor nerve innervating it with a single stimulus causes a single contraction of the muscle. It distinguishes two main phases: the contraction phase and the relaxation phase. Contraction of the muscle fiber begins already during the ascending branch of the action potential. The duration of contraction at each point of the muscle fiber is tens of times longer than the duration of the AP. Therefore, there comes a moment when the AP has passed along the entire fiber and has ended, but the wave of contraction has engulfed the entire fiber and it continues to be shortened. This corresponds to the moment of maximum shortening or tension of the muscle fiber.

The contraction of each individual muscle fiber during single contractions obeys the law " all or nothing". This means that the contraction that occurs during both threshold and superthreshold stimulation has a maximum amplitude. The magnitude of a single contraction of the entire muscle depends on the strength of the stimulation. With threshold stimulation, its contraction is barely noticeable, but with increasing strength of stimulation it increases, until it reaches a certain height, after which it remains unchanged (maximum contraction). This is explained by the fact that the excitability of individual muscle fibers is not the same, and therefore only part of them is excited with weak stimulation. With maximum contraction, they are all excited. The speed of the muscle contraction wave is the same with the speed of propagation of the action force. In the biceps brachii muscle it is 3.5-5.0 m/sec.

Single contraction - reduction by one stimulus. It is divided into a latent period, a contraction phase and a relaxation phase. At the moment of the latent period, the refractory phase occurs. But already at the beginning of the shortening phase it is restored.

12. Summation of muscle contractions. Tetanic contractions.

If in an experiment two strong single stimulations act on a single muscle fiber or the entire muscle in rapid succession, the resulting contraction will have a greater amplitude than the maximum single contraction. The contractile effects caused by the first and second stimulation seem to add up. This phenomenon is called summation of contractions. For summation to occur, it is necessary that the interval between irritations have a certain duration - it must be longer than the refractory period, but shorter than the entire duration of a single contraction, so that the second irritation affects the muscle before it has time to relax. In this case, two cases are possible. If the second stimulus arrives when the muscle has already begun to relax, on the myographic curve the apex of the second contraction will be separated from the first by a retraction. If the second stimulation acts when the first contraction has not yet reached its peak, then the second contraction seems to merge with the first, forming together with it a single summed peak. For both full and incomplete summation, PDs are not summed up. This summed contraction in response to rhythmic stimulation is called tetanus. Depending on the frequency of irritation, it can be jagged or smooth.

The reason for the summation of contractions during tetanus lies in the accumulation of Ca++ ions in the interfibrillar space to a concentration of 5*10 6 mmol/l. After reaching this value, further accumulation of Ca++ does not lead to an increase in the amplitude of tetanus.

After the cessation of tetanic stimulation, the fibers do not relax completely at first, and their original length is restored only after some time. This phenomenon is called post-tetanic, or residual contracture. It's related to that. that it takes more time to remove from the interfibrillar space all the Ca++ that got there during rhythmic stimuli and did not have time to be completely removed into the cisterns of the sarcoplasmic reticulum by the work of Ca pumps.

If, after achieving smooth tetanus, the frequency of stimulation is increased even more, then the muscle at a certain frequency suddenly begins to relax. This phenomenon is called pessimum . It occurs when each subsequent impulse falls into refractoriness from the previous one.

13. Ultrastructure of myofibrils. Contractile proteins (actin, myosin). Regulatory proteins (troponin, tropomyosin) in the composition of thin protofibrils. The theory of muscle contraction.

Myofibrils are the contractile apparatus of muscle fibers. In striated muscle fibers, myofibrils are divided into regularly alternating sections (discs) that have different optical properties. Some of these areas are anisotropic, i.e. are birefringent. In normal light they appear dark, but in polarized light they appear transparent in the longitudinal direction and opaque in the transverse direction. Other areas are isotropic and appear transparent in normal light. Anisotropic areas are designated by the letter A, isotropic - I. There is a light stripe in the middle of disk A N, and in the middle of disk I there is a dark stripe Z, which is a thin transverse membrane through the pores of which myofibrils pass. Due to the presence of such a supporting structure, the parallel, single-digit discs of individual myofibrils within one fiber do not move relative to each other during contraction.

It has been established that each of the myofibrils has a diameter of about 1 μm and consists of an average of 2500 protofibrils, which are elongated polymerized molecules of myosin and actin proteins. Myosin filaments (protofibrils) are twice as thick as actin filaments. Their diameter is approximately 100 angstroms. In the resting state of the muscle fiber, the filaments are located in the myofibril in such a way that thin long actin filaments enter their ends into the spaces between thick and shorter myosin filaments. In such a section, each thick thread is surrounded by 6 thin ones. Due to this, disks I consist only of actin filaments, and disks A also consist of myosin filaments. The light stripe H represents a zone free of actin filaments during the resting period. Membrane Z, passing through the middle of disk I, holds actin filaments together.

An important component of the ultramicroscopic structure of myofibrils are also numerous cross-bridges on myosin. In turn, actin filaments have so-called active centers, which at rest are covered, like a cover, by special proteins - troponin and tropomyosin. Contraction is based on the process of sliding of actin filaments relative to myosin filaments. This sliding is caused by the work of the so-called. "chemical gear", i.e. periodically occurring cycles of changes in the state of cross bridges and their interaction with active centers on actin. ATP and Ca+ ions play an important role in these processes.

When a muscle fiber contracts, the actin and myosin filaments do not shorten, but begin to slide over each other: the actin filaments move between the myosin filaments, as a result of which the length of the I disks is shortened, and the A disks maintain their size, moving closer to each other. The H strip almost disappears because the ends of actin touch and even overlap each other.

14. Relationship between excitation and contraction (electromechanical coupling) in muscle fibers. The role of calcium ions. Function of the sarcoplasmic reticulum.

In skeletal muscle under natural conditions, the initiator of muscle contraction is the action potential, which, when excited, propagates along the surface membrane of the muscle fiber.

If the tip of the microelectrode is applied to the surface of the muscle fiber in the region of membrane Z, then when a very weak electrical stimulus is applied that causes depolarization, disks I on both sides of the site of stimulation will begin to shorten. in this case, the excitation spreads deep into the fiber, along the membrane Z. Irritation of other parts of the membrane does not cause such an effect. It follows from this that depolarization of the surface membrane in the area of ​​disk I during AP propagation is the triggering mechanism for the contractile process.

Further studies showed that an important intermediate link between membrane depolarization and the onset of muscle contraction is the penetration of free CA++ ions into the interfibrillar space. At rest, the majority of Ca++ in muscle fiber is stored in the sarcoplasmic reticulum.

In the mechanism of muscle contraction, a special role is played by that part of the reticulum that is localized in the region of membrane Z. Electron microscopy reveals the so-called reticulum. triad (T-system), each of which consists of a thin transverse tube centrally located in the region of the membrane Z, running across the fiber, and two lateral cisterns of the sarcoplasmic reticulum, which contain bound Ca++. The PD, spreading along the surface membrane, is carried deep into the fiber along the transverse tubes of the triads. Then the excitation is transmitted to the cisterns, depolarizes their membrane and it becomes permeable to CA++.

It has been experimentally established that there is a certain critical concentration of free Ca++ ions at which contraction of myofibrils begins. It is equal to 0.2-1.5 * 10 6 ions per fiber. An increase in Ca++ concentration to 5*10 6 already causes a maximum contraction.

The onset of muscle contraction is confined to the first third of the ascending limb of the AP, when its value reaches approximately 50 mV. It is believed that it is at this magnitude of depolarization that the Ca++ concentration becomes the threshold for the onset of interaction between actin and myosin.

The process of Ca++ release stops after the end of the AP peak. Nevertheless, the contraction continues to increase until the mechanism that ensures the return of Ca++ to the reticulum cisterns comes into play. This mechanism is called the “calcium pump”. To carry out its work, the energy obtained from the breakdown of ATP is used.

In the interfibrillar space, Ca++ interacts with proteins that cover the active centers of actin filaments - troponin and tropomyosin, providing the opportunity for the reaction of myosin cross bridges and actin filaments.

Thus, the sequence of events leading to contraction and then relaxation of the muscle fiber is currently depicted as follows:

15. Fatigue during muscular work. Causes of fatigue. The concept of active recreation.

Fatigue is a temporary decrease in the performance of a cell, organ or entire organism that occurs as a result of work and disappears after rest.

If you irritate an isolated muscle for a long time with rhythmic electrical stimuli, to which a small load is suspended, then the amplitude of its contractions gradually decreases until it drops to zero. A fatigue curve is recorded. Along with a change in the amplitude of contractions during fatigue, the latent period of contraction increases, the period of muscle relaxation lengthens and the threshold of irritation increases, i.e. excitability decreases. All these changes do not occur immediately after the start of work; there is a certain period during which there is an increase in the amplitude of contractions and a slight increase in muscle excitability. At the same time, it becomes easily stretchable. In such cases, they say that the muscle is “worked in,” i.e. adapts to work in a given rhythm and strength of irritation. After a period of workability, a period of stable performance begins. With further prolonged irritation, muscle fiber fatigue occurs.

The decrease in the performance of a muscle isolated from the body during prolonged irritation is due to two main reasons. The first of them is that during contractions, metabolic products accumulate in the muscle (phosphoric acid, Ca++ binding, lactic acid, etc.), which have a depressing effect on muscle performance. Some of these products, as well as Ca ions, diffuse from the fibers out into the pericellular space and have a suppressive effect on the ability of the excitable membrane to generate AP. So, if an isolated muscle placed in a small volume of Ringer’s fluid is brought to the point of complete fatigue, then it is enough just to change the solution washing it to restore muscle contractions.

Another reason for the development of fatigue in an isolated muscle is the gradual depletion of its energy reserves. With prolonged work, the glycogen content in the muscle sharply decreases, as a result of which the processes of resynthesis of ATP and CP necessary for contraction are disrupted.

It should be noted that under the natural conditions of the organism’s existence, fatigue of the motor system during prolonged work develops completely differently than in an experiment with an isolated muscle. This is due not only to the fact that in the body the muscle is continuously supplied with blood, and, therefore, receives the necessary nutrients with it and is freed from metabolic products. The main difference is that in the body, exciting impulses come to the muscle from the nerve. The neuromuscular synapse gets tired much earlier than the muscle fiber, due to the rapid depletion of the accumulated neurotransmitter reserves. This causes a blockade of the transmission of excitations from the nerve to the muscle, which protects the muscle from exhaustion caused by prolonged work. In the whole organism, the nerve centers (nervous-nervous contacts) become tired even earlier during work.

The role of the nervous system in the fatigue of the entire organism is proven by studies of fatigue in hypnosis (weight-basket), establishing the influence of “active rest” on fatigue, the role of the sympathetic nervous system (Orbeli-Ginetzinsky phenomenon), etc.

Ergography is used to study muscle fatigue in humans. The shape of the fatigue curve and the amount of work performed varies extremely among different individuals and even among the same subject under different conditions.

16. Physiological characteristics of smooth muscles. Plastic tone of smooth muscles.

An important property of smooth muscle is its large plastic , those. the ability to maintain the length given by stretching without changing the tension. Skeletal muscle, on the contrary, immediately shortens after the load is removed. The smooth muscle remains stretched until, under the influence of some irritation, its active contraction occurs. The property of plasticity is of great importance for the normal functioning of hollow organs - thanks to it, the pressure inside a hollow organ changes relatively little with different degrees of its filling.

There are different types of smooth muscles. In the walls of most hollow organs there are muscle fibers with a length of 50-200 microns and a diameter of 4-8 microns, which are very closely adjacent to each other, and therefore, when examined under a microscope, it seems that they morphologically form one whole. Electron microscopic examination shows, however, that they are separated from each other by intercellular gaps, the width of which can be 600-1500 angstroms. Despite this, smooth muscle functions as one unit. This is expressed in the fact that AP and slow waves of depolarization propagate unhindered from one fiber to another.

In some smooth muscles, for example, in the ciliary muscle of the eye, or the muscles of the iris, the fibers are located separately, and each has its own innervation. In most smooth muscles, motor nerve fibers are located on only a small number of fibers.

The resting potential of smooth muscle fibers, which have automaticity, exhibits constant small fluctuations. Its value during intracellular abduction is 30-70 mV. The resting potential of smooth muscle fibers that do not have automaticity is stable and equal to 60-70 mV. In both cases, its value is less than the resting potential of skeletal muscle. This is due to the fact that the membrane of smooth muscle fibers at rest is characterized by a relatively high permeability to Na ions. Action potentials in smooth muscles are also slightly lower than in skeletal muscles. The excess over the resting potential is no more than 10-20 mV.

The ionic mechanism of AP occurrence in smooth muscles is somewhat different from that in skeletal muscles. It has been established that regenerative membrane depolarization, which underlies the action potential in a number of smooth muscles, is associated with an increase in membrane permeability for Ca++ ions, rather than Na+.

Many smooth muscles exhibit spontaneous, automatic activity. It is characterized by a slow decrease in the resting membrane potential, which, when a certain level is reached, is accompanied by the occurrence of AP.

nerve and skeletal muscle fibers, excitation spreads through local electrical currents arising between the depolarized and adjacent resting sections of the cell membrane. The same mechanism is also characteristic of smooth muscles. However, unlike what occurs in skeletal muscles, in smooth muscles an action potential arising in one fiber can spread to adjacent fibers. This is due to the fact that in the membrane of smooth muscle cells in the area of ​​​​contacts with neighboring ones there are areas of relatively low resistance, through which current loops that arise in one fiber easily pass to neighboring ones, causing depolarization of their membranes. In this respect, smooth muscle is similar to cardiac muscle. The only difference is that in the heart, the entire muscle is excited from one cell, and in smooth muscles, the PD that arises in one area spreads from it only to a certain distance, which depends on the strength of the applied stimulus.

Another significant feature of smooth muscles is that a spreading action potential occurs downward only if the applied stimulus simultaneously excites a certain minimum number of muscle cells. This "critical zone" has a diameter of about 100 microns, which corresponds to 20-30 parallel cells. The speed of excitation in various smooth muscles ranges from 2 to 15 cm/sec. those. significantly less than in skeletal muscle.

Just as in skeletal muscles, in smooth muscles action potentials have a trigger value for the onset of the contractile process. The connection between excitation and contraction here is also carried out with the help of Ca++. However, in smooth muscle fibers the sarcoplasmic reticulum is poorly expressed, so the leading role in the mechanism of contraction is assigned to those Ca++ ions that penetrate into the muscle fiber during the generation of action potential.

With a large force of single irritation, contraction of the smooth muscle may occur. The latent period of its contraction is much longer than the skeletal one, reaching 0.25-1 sec. The duration of the contraction itself is also long - up to 1 minute. Relaxation occurs especially slowly after contraction. The contraction wave propagates through the smooth muscles at the same speed as the excitation wave (2-15 cm/sec). But this slowness of contractile activity is combined with great force of smooth muscle contraction. Thus, the muscles of the stomach of birds are capable of lifting 2 kg per 1 sq. mm. its cross section.

Due to the slowness of contraction, smooth muscle, even with rare rhythmic stimulation (10-12 per minute), easily goes into a long-term state of persistent contraction, reminiscent of skeletal muscle tetanus. However, the energy costs for such a reduction are very low.

The ability to automate smooth muscles is inherent in their muscle fibers and is regulated by nerve elements that are located in the walls of smooth muscle organs. The myogenic nature of automaticity has been proven by experiments on strips of intestinal wall muscles freed from nervous elements. Smooth muscle reacts to all external influences by changing the frequency of spontaneous rhythms, resulting in contractions or relaxations of the muscle. The effect of irritation of intestinal smooth muscles depends on the relationship between the frequency of stimulation and the natural frequency of spontaneous rhythms: with low tone - rare spontaneous PD - the applied irritation increases the tone; with high tone, relaxation occurs in response to irritation, since excessive acceleration of impulses leads to each subsequent impulse falls into a refractory phase from the previous one.

17. Structure and functions of nerve fibers. The mechanism of excitation

myelinated and unmyelinated nerve fibers. The meaning of nodes of Ranvier.

The main function of axons is to conduct impulses arising in a neuron. Axons may be covered with a myelin sheath (myelinated fibers) or lack it (unmyelinated fibers). Myelinated fibers are more common in motor nerves, while non-myelinated fibers predominate in the autonomic (autonomic) nervous system.

An individual myelinated nerve fiber consists of an axial cylinder covered by a myelin sheath formed by Schwann cells. The axial cylinder has a membrane and axoplasm. The myelin sheath is a product of the activity of the Schwann cell and consists of 80% lipids with high ohmic resistance and 20% protein.

The myelin sheath does not cover the axial cylinder with a continuous cover, but is interrupted, leaving open areas of the axial cylinder, called nodes of Ranvier. The length of the sections between these interceptions is different and depends on the thickness of the nerve fiber: the thicker it is, the longer the distance between the interceptions (Fig. 2.17).

Unmyelinated nerve fibers are covered only by Schwann's sheath.

The conduction of excitation in unmyelinated fibers differs from that in myelinated fibers due to the different structure of the membranes. In unmyelinated fibers, excitation gradually covers adjacent sections of the membrane of the axial cylinder and thus spreads to the end of the axon. The speed of excitation propagation along the fiber is determined by its diameter.

In nerve fibers without myelin, where metabolic processes do not provide rapid compensation for energy expenditure on excitation, the spread of this excitation occurs with a gradual weakening - with decrement. Decremental conduction of excitation is characteristic of a low-organized nervous system.

In higher animals, thanks primarily to the presence of the myelin sheath and the perfection of metabolism in the nerve fiber, excitation passes without fading, without decrement. This is facilitated by the presence of an equal charge throughout the fiber membrane and its rapid restoration after the passage of excitation.

In myelinated fibers, excitation covers only areas of nodal interceptions, that is, it bypasses areas covered with myelin. This conduction of excitation along the fiber is called saltatory (spacing). In the nodes, the number of sodium channels reaches 12,000 per 1 µm, which is significantly more than in any other part of the fiber. As a result, the nodal interceptions are the most excitable and provide a greater speed of excitation. The conduction time of excitation along the myelin fiber is inversely proportional to the length between interceptions.

The conduction of excitation along the nerve fiber is not disrupted for a long time (many hours). This indicates low fatigue of the nerve fiber. It is believed that the nerve fiber is relatively tireless due to the fact that the processes of energy resynthesis in it proceed at a sufficiently high speed and manage to restore the energy expenditure that occurs during the passage of excitation.

At the moment of excitation, the energy of the nerve fiber is spent on the operation of the sodium-potassium pump. Particularly large amounts of energy are wasted at the nodes of Ranvier due to the high density of sodium-potassium channels here.

J. Erlanger and H. Gasser (1937) were the first to classify nerve fibers based on the speed of excitation. The speed of excitation along the mixed nerve fibers varies when using an extracellular electrode. The potentials of fibers conducting excitation at different speeds are recorded separately (Fig. 2.18).

Depending on the speed of excitation, nerve fibers are divided into three types: A, B, C. In turn, type A fibers are divided into four groups: A α , A β , A γ , A δ . Group A fibers have the highest conduction speed (up to 120 m/s) α , which consists of fibers with a diameter of 12-22 microns. Other fibers have a smaller diameter and, accordingly, excitation through them occurs at a lower speed (Table 2.4).

The nerve trunk is formed by a large number of fibers, but the excitation going along each of them is not transmitted to the neighboring ones. This feature of the conduction of excitation along the nerve is called law of isolated excitation conduction along a single nerve fiber. The possibility of such conduct is of great physiological importance, since it ensures, for example, the isolation of the contraction of each neuromotor unit.

The ability of a nerve fiber to conduct excitation in an isolated manner is due to the presence of membranes, as well as the fact that the resistance of the fluid filling the interfiber spaces is significantly lower than the resistance of the fiber membrane. Therefore, the current, leaving the excited fiber, is shunted in the liquid and turns out to be weak for exciting neighboring fibers. A necessary condition for the conduction of excitation in a nerve is not just its anatomical continuity, but also its physiological integrity. In any metal conductor, electric current will flow as long as the conductor maintains physical continuity. For a nerve “conductor” this condition is not enough: the nerve fiber must also maintain physiological integrity. If the properties of the fiber membrane are violated (ligation, blockade with novocaine, ammonia, etc.), the conduction of excitation along the fiber stops. Another property characteristic of the conduction of excitation along a nerve fiber is the ability for bilateral conduction. Applying stimulation between two output electrodes on the surface of a fiber will induce electrical potentials beneath each electrode.

Table - Velocity of excitation along nerve fibers

Fiber group	Fiber diameter, µm	Conduction speed, m/s

18. Laws of conduction of excitation along the nerves. Classification of nerve fibers. The speed of excitation along nerve fibers, its age-related characteristics.

19. Structure of the neuromuscular synapse. The mechanism of transmission of excitation from nerve to muscle.End plate potential, its properties.

Synapses are the contacts that establish neurons as independent entities. The synapse is a complex structure and consists of a presynaptic part (the end of the axon that transmits the signal), a synaptic cleft and a postsynaptic part (the structure of the receiving cell).

Neuromuscular synapses ensure the conduction of excitation from the nerve fiber to the muscle fiber thanks to the mediator acetylcholine, which, when the nerve ending is excited, passes into the synaptic cleft and acts on the end plate of the muscle fiber.

Acetylcholine is formed and accumulates in the form of vesicles in the presynaptic terminal. When excited by an electrical impulse traveling along the axon, the presynaptic part of the synapse becomes permeable to acetylcholine.

This permeability is possible due to the fact that as a result of depolarization of the presynaptic membrane, its calcium channels open. The Ca2+ ion enters the presynaptic part of the synapse from the synaptic cleft. Acetylcholine is released and enters the synaptic cleft. Here it interacts with its receptors on the postsynaptic membrane belonging to the muscle fiber. The receptors, when excited, open a protein channel embedded in the lipid layer of the membrane. Na+ ions penetrate into the muscle cell through the open channel, which leads to depolarization of the muscle cell membrane, resulting in the development of the so-called end plate potential (EPP). Since this potential is normally always above threshold, it causes an action potential that propagates along the muscle fiber and causes contraction. The end plate potential is short, since acetylcholine, firstly, is quickly disconnected from the receptors, and secondly, it is hydrolyzed by AChE.

The neuromuscular synapse transmits excitation in one direction: from the nerve ending to the postsynaptic membrane of the muscle fiber, which is due to the presence of a chemical link in the mechanism of neuromuscular transmission.

The speed of excitation through the synapse is much less than along the nerve fiber, since time is spent here on the activation of the presynaptic membrane, the passage of calcium through it, the release of acetylcholine into the synaptic cleft, the depolarization of the postsynaptic membrane, and the development of PPP.

Explanatory note

The proposed elective course contains information about the cell - the elementary unit of living nature - and is intended for students of specialized classes with an interest in cytology and biochemistry. The proposed elective course supports and deepens basic knowledge of biology. Studying an elective course will help in choosing further education and professional activities.

The course builds on the knowledge and skills acquired by students while studying biology. During the lessons, students are expected to gain experience in searching for information on the proposed issues. Students improve their skills in preparing abstracts, reports, messages on a chosen topic, and practice experimental techniques.

The elective course lasts 35 hours. The program provides for the study of theoretical issues, conducting seminars and laboratory work.

Purpose of the course: in-depth study of the structure and properties of the cell, necessary for a comprehensive understanding of biological facts, phenomena and processes.

Course objectives: developing the ability to identify, reveal, and use the relationship between the structure and function of a cell when considering biological processes and phenomena; consolidation of skills and abilities necessary for laboratory work; involving students in independent work with additional literature; stimulating the cognitive activity of students interested in cytology and biochemistry; formation of skills and abilities for a comprehensive understanding of knowledge in biology.

Basic concept of the course: the use of an integrated approach when studying organisms at different levels of organization, the formation of evolutionary thinking.

As a result of studying the course students should know:

– device of a microscope and methods of working with it;
– provisions of the cell theory;
– similarities and differences between plant and animal cells;
– the role of various chemical compounds in the cell;
– main components and organelles of the cell;
– structural features of prokaryotic and eukaryotic cells;
– disorders of protein and carbohydrate metabolism;
– the significance of individual mineral elements.

Students should be able to:

– work with a microscope;
– name the main parts of the cell, recognize them in diagrams and photographs;
– make simple preparations for microscopic examination;
– correctly format laboratory work;
– independently work with additional literature and use modern technologies.

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Topic 1. Cell: history of study (3 hours)

Lesson #1. Introduction to cell cytology. A cell is an integral system. History of the study of cells. Tasks of modern cytology.

Lesson #2 . Creation of cell theory. Methods for studying cells. Parallelism in the evolution of microscopic technology and the level of cytological research.

Lesson #3 . Laboratory work No. 1."Microscope design and microscopy techniques."

Topic 2. Cell chemistry (8 hours)

Lesson #1. Chemical elements in the cell. Features of the chemical composition of living things. Ions in the cell and body. The content of chemical compounds in the cell. The role of water in a living system.

Lesson #2 . Organic compounds. Chemistry of proteins. Proteins are colloids, proteins are amphoteric electrolytes, proteins are hydrophilic compounds.

Laboratory work No. 2."Evidence for the biocatalytic function of enzyme proteins".

Lesson #3 . Essential amino acids. Pathological phenomena in the absence of proteins in food and disorders of protein metabolism.

Lesson #4 . Laboratory work No. 3."Detection of proteins in biological objects."

Lesson #5 . Carbohydrates are the most common organic substances on Earth. Relationship between the structure of carbohydrates and biological functions. Pathologies associated with impaired carbohydrate metabolism in the body: blood sugar levels are normal and in pathologies, hyper- and hypoglycemia, diabetes mellitus.

Lesson #6 . Laboratory work No. 4."Detection of carbohydrates in biological objects."

Lesson #7 . Lipids. The role of lipids in the emergence of a certain autonomy of a biological system such as a cell.

Laboratory work No. 5."Detection of lipids in biological objects."

Lesson #8 . Nucleic acids. Watson and Crick model.

Laboratory work No. 6.“Isolation of deoxynucleoprotein from spleen (liver) tissue. Qualitative reaction to DNA."

Topic 3. General plan of the cell structure (10 hours)

Lesson #1 . Membrane. Modern model of the structure of the cell membrane. Cell wall, glycocalyx.

Lesson #2 . Cytoskeleton, its components and functions in different types of cells.

Lesson #3 . Membrane transport.

Lesson #4 . Endocytosis and receptor function of the membrane.

Lessons No. 5–6 . Membrane organelles of the cell.

Lessons No. 7–8. Non-membrane cell organelles. Prokaryotes and eukaryotes. Animal and plant eukaryotic cell.

Laboratory work No. 7."Structural features of prokaryotes and eukaryotes."

Lesson #9 . Laboratory work No. 8."Physiological properties of the cell membrane".

Lesson #10 . Seminar. Prospects for the development of membranology.

Topic 4. Metabolism (6 hours)

Lesson #1 . Sources of cell energy. Heterotrophs and autotrophs.

Lesson #2 . Mitochondria are the energy stations of the cell. ATP biosynthesis scheme.

Lesson #3 . The mechanism of photosynthesis. Chemosynthesis.

Lesson #4 . Biosynthesis of proteins. Gene structure. Transcription.

Lesson #5 . Ribosomes. Types and structures of ribosomes in prokaryotes and eukaryotes.

Lesson #6 . Broadcast. Regulation of transcription and translation. Epigenetic factors.

Topic 5. Nuclear apparatus and cell reproduction (6 hours)

Lesson #1 . Concept of chromatin. The nucleus of a eukaryotic cell. Karyoplasm.

Lesson #2 . Life cycle of a cell. Cell reproduction.

Lesson #3 . Concept of stem cells.

Lesson #4 . Aging and cell death. Necrosis and apoptosis. Cancer.

Lesson #5 . Laboratory work No. 9. "Mitosis in onion root cells."

Lesson #6 . Seminar.

Topic 6. Cell evolution (2 hours)

Lessons #1–2 . Theory of evolution of prokaryotes and eukaryotes. Final conference “Primary stages of biological evolution”.

Laboratory work No. 1. “The design of a light microscope and microscopy techniques”

Goal of the work: based on knowledge of the structure of a light microscope, master the technique of microscopy and preparation of temporary microslides. Familiarize yourself with the rules for completing laboratory work.

Equipment: microscope for each student. Slides and cover glasses, pipettes, cups of water, cotton wool, filter paper, tweezers, scissors, notebook, album. Diagram of the microscope and its parts.

Progress

Consider the main parts of a microscope: mechanical, optical and lighting.

The mechanical part includes: a tripod, a stage, a tube, a revolver, macro- and micrometer screws.

The optical part of the microscope is represented by an eyepiece and objectives. Eyepiece (lat. okulus– eye) is located in the upper part of the tube and faces the eye. This is a system of lenses enclosed in a sleeve. By the number on the upper surface of the eyepiece you can judge its magnification factor (×7, ×10, ×15). If necessary, the eyepiece can be removed from the tube and replaced with another. On the opposite side of the tube is a rotating plate, or revolver (lat. revolvo– rotate), which contains lens sockets. A lens is a system of lenses, they have different magnifications. There is a low magnification lens (×8), a high magnification lens (×40) and an immersion lens for studying small objects (×90). The total magnification of the microscope is equal to the magnification of the eyepiece multiplied by the magnification of the objective.

The lighting part consists of a mirror, a condenser and a diaphragm.

The condenser is located between the mirror and the stage. It consists of two lenses. A special screw is used to move the condenser. When the condenser is lowered, the illumination decreases; when it is raised, it increases. By changing the position of the diaphragm plates, you can also adjust the lighting using a special knob.

Exercise: Draw a microscope and label its parts.

Rules for working with a microscope

1. Set up the microscope with the tripod facing you and the stage away from you.

2. Place the low magnification lens in working position.

3. While looking through the eyepiece with your left eye, rotate the mirror in different directions until the field of view is brightly and evenly illuminated.

4. Place the prepared preparation on the stage (cover glass up) so that it is in the center of the hole in the stage.

5. Under visual control (look not through the eyepiece, but from the side), slowly lower the tube using the macroscrew so that the lens is at a distance of 2 mm from the specimen.

6. Looking through the eyepiece, slowly lift the tube until an image of the object appears.

7. In order to proceed to examining the object at high magnification of the microscope, it is necessary to center the preparation, i.e. place the object in the center of the field of view.

8. Rotating the revolver, move the high-magnification lens to the working position.

9. Lower the tube under visual control almost until it comes into contact with the drug.

10. Looking through the eyepiece, slowly lift the tube until an image appears.

11. For fine focusing, use a microscopic screw.

12. When sketching the preparation, look into the eyepiece with your left eye.

Exercise: rewrite the rules for working with a microscope in a notebook for laboratory work.

Method for preparing a temporary preparation

1. Take a glass slide, holding it by its side edges, and place it on the table.

2. Place an object, for example 1.5 cm long pieces of cotton wool, in the center of the glass. Using a pipette, apply one drop of water to the object.

3. Place a cover glass on the slide. Remove excess water with a piece of filter paper.

4. Consider the finished drug.

5. Sketch in an album what the cotton wool fibers look like at low and high magnification.

Microscoping of protozoa

From an aquarium that has not been cleaned for a long time, take a drop along with a plant twig or duckweed leaf and examine it under a microscope at low magnification. Usually a variety of protozoa are visible: slipper ciliates, amoebas - free-living and attached to algae (suvoiki). There may also be multicellular organisms in the water - small worms and crustaceans (cyclops, daphnia). By looking at this preparation, you can practice pointing the microscope at moving objects.

Rules for completing laboratory work

A necessary element of microscopic study of an object is its sketching in an album. To do this, you need to have a 21x30 cm album and pencils (simple and colored). A notebook is needed to record text material and complete diagrams.

1. You can only draw on one side of the sheet.

2. Before starting the sketch, write down the name of the topic at the top of the page.

3. The drawing should be large, the details are clearly visible.

4. The drawing must correctly reflect the shapes, the ratio of volume and size of individual parts and the whole.

First you need to draw the outline of the object (large), then inside - the outlines of the details and after that clearly draw them.

5. Draw, clearly repeating all the lines of the object. To do this, you must not take your eyes off the microscope, but only switch your attention from the object to the drawing (you need to learn this).

6. Each drawing must be labeled with parts. All inscriptions must be parallel to each other. Arrows are placed on individual parts of the object, and the name of the part of the object is written against each part.

Laboratory work No. 2. “Proof of the biocatalytic function of enzyme proteins”

Goal of the work: prove the catalytic effect of enzyme proteins, show their high specificity, optimal activity under physiological conditions.

Equipment: rack with test tubes, 1 ml pipettes, water bath, thermostat; 1% starch solution, 1% iodine solution in potassium iodide, 5% copper sulfate solution, 10% sodium hydroxide solution, 2% sucrose solution, 0.2% hydrochloric acid solution, saliva solution diluted with water 5 times (to 1 volume saliva add 4 volumes of water).

Progress

1. Enzymatic hydrolysis of starch

Salivary amylase acts as an enzyme that hydrolyzes starch into its constituent parts (maltose, glucose). The results of the experiment are assessed using color reactions - with iodine and the Trommer reaction (qualitative reaction to glucose). Unhydrolyzed starch gives a blue color with iodine and a negative Trommer reaction. The products of starch hydrolysis do not react with iodine, but give color in the Trommer reaction.

Volumes can be measured in drops: 1 drop is approximately 0.2 ml.

1. Take two test tubes (No. 1 and No. 2) and add 10 drops of 1% starch solution to each.

2. Add 4 drops of water (control) to test tube No. 1, mix the contents carefully and place the test tube in a water bath or in a thermostat at 37 °C for 20 minutes.

3. After 5 minutes, add 4 drops of diluted saliva solution into test tube No. 2 and also place in the thermostat for 20 minutes,

4. After keeping in a thermostat, transfer 4 drops from test tube No. 1 to 2 different test tubes.

5. Add 1 drop of a 1% solution of iodine in potassium iodide to one of the test tubes, 1 drop of a 5% solution of copper sulfate and 4 drops of a 10% solution of sodium hydroxide to the other, and carefully heat this test tube to a boil.

6. Repeat the same with the contents of test tube No. 2.

In the presence of water, hydrolysis of starch does not occur, and in the reaction with iodine a blue color of starch should appear, and in the Trommer reaction the solution should remain blue. Salivary amylase hydrolyzes starch to glucose: there is no reaction with iodine, but in the Trommer reaction, coloration occurs first yellow (formation of CuOH), and then brick-red (formation of Cu(OH) 2).

2. Specificity of enzyme action

Each enzyme acts on only one substance or group of similar substrates. This is due to the correspondence between the structure of the enzyme (its active center) and the structure of the substrate. For example, amylase acts only on starch, and sucrase only on sucrose.

1. Preparation of sucrase solution. Take 10 g of fresh or 3 g of dry baker's yeast, grind in a porcelain mortar with 6 ml of distilled water, add 20 ml of water and filter through cotton wool (store in the refrigerator).

2. Preparation of amylase solution. Measure out 50 ml of distilled water and rinse your mouth with it in 2-4 doses for 3-5 minutes. The collected liquid is filtered through cotton wool and used as an amylase solution.

3. Take 4 test tubes. Add 10 drops of 1% starch solution into test tubes No. 1 and No. 2. Add 10 drops of 2% sucrose solution to test tubes No. 3 and No. 4. Add 5 drops of amylase solution to test tubes No. 1 and No. 3. Add 5 drops of sucrase solution to test tubes No. 2 and No. 4. Stir and keep in a thermostat at a temperature of 38–40 °C for 15 minutes.

Carry out reactions with iodine and Trommer with the contents of all four test tubes. Fill out the table.

Table. Determination of the specificity of enzyme action

In the conclusions, it should be noted in which test tube and under what conditions the action of the enzymes was detected and why.

3. Effect of pH on enzyme activity

For each enzyme there is a certain reaction value of the environment at which it exhibits maximum activity. A change in the pH of the environment causes a decrease or complete inhibition of enzyme activity.

Add 1 ml of distilled water to 8 test tubes. Add 1 ml of 0.2% hydrochloric acid solution to test tube No. 1 and mix. Take 1 ml of the mixture from test tube No. 1 and transfer it to test tube No. 2, mix, transfer 1 ml to test tube No. 3, etc. Take 1 ml from test tube No. 8 and pour it away. We obtain media with different pH values. pH values ​​can be checked with a pH meter or universal indicator paper.

Add 2 ml of 1% starch solution and 1 ml of amylase solution to each test tube (see above). Shake the test tubes and place in a thermostat at 37 ° C for 15 min.

After cooling, add one drop of a 1% solution of iodine in potassium iodide to all test tubes. Complete hydrolysis will occur in test tubes No. 5 and No. 6, where the pH of the solution medium is in the range of 6.8–7.2, i.e. under optimal conditions for amylase action.

Laboratory work No. 3. “Detection of proteins in biological objects”

Goal of the work: prove the presence of proteins in biological objects.

Equipment: rack with test tubes, pipette, water bath, dropper; egg white solution; 10% NaOH solution, 1% copper sulfate solution, 0.5% aqueous ninhydrin solution; nitric acid (concentrated).

Progress

1. Biuret reaction for determining peptide bonds.

The method is based on the ability of a peptide bond in an alkaline environment to form colored complex compounds with copper sulfate.

Add 5 drops of a 10% egg white solution (the white is filtered through gauze, then diluted 1:10 with distilled water), three drops of a 10% sodium hydroxide solution and 1 drop of a 1% copper sulfate solution into a test tube and mix.

The contents of the test tube acquire a blue-violet color.

2. Ninhydrin reaction.

Proteins, polypeptides and free amino acids give a blue or violet color with ninhydrin.

Add 5 drops of a 10% egg white solution into a test tube, add 5 drops of a 0.5% aqueous solution of ninhydrin and heat. After 2–3 minutes, a pink or blue-violet color develops.

3. Xanthoprotein reaction (Greek. xantos- yellow). Using this reaction, amino acids containing benzene rings (tryptophan, tyrosine, etc.) are detected in the protein.

Add 5 drops of a 10% egg white solution into a test tube, add 3 drops of concentrated nitric acid (carefully) and heat. After cooling, add 5–10 drops of 10% sodium hydroxide solution into the test tube until an orange color appears (this is associated with the formation of sodium salt of cyclic nitro compounds).

Crystals in plant cells

Laboratory work No. 4. “Detection of carbohydrates in biological objects”

Goal of the work: prove the presence of carbohydrates in biological objects.

Equipment: rack with test tubes; pipettes, water bath; 1% starch solution, 1% sucrose solution, 1% fructose solution, 1% iodine solution (in potassium iodide solution); 1% alcohol solution of naphthol, 1% alcohol solution of thymol; sulfuric acid (concentrated); Selivanov's reagent (0.5 g of resorcinol in 100 ml of 20% hydrochloric acid).

Progress

1. Detection of starch.

Add 10 drops of a 1% starch solution and one drop of a 1% solution of iodine in potassium iodide into a test tube. The contents of the test tube acquire a blue-violet color.

2. Detection of carbohydrates.

By reaction with naphthol or thymol, small amounts of carbohydrates or carbohydrate components in complex compounds are detected.

Add 10 drops of 1% sucrose solution into two test tubes. Add 3 drops of a 1% alcohol solution of naphthol to one test tube. In another test tube - 3 drops of a 1% alcohol solution of thymol. Pour 0.5 ml of concentrated sulfuric acid into both (carefully) and at the border of the two liquids observe a violet color in a test tube with naphthol and a red color in a test tube with thymol.

3. Detection of fructose (Selivanov reaction).

Fructose, when heated with hydrochloric acid and resorcinol, gives a cherry-red color.

Pour 10 drops of Selivanov’s reagent and 2 drops of 1% fructose solution into a test tube and heat gently. A red color is observed.

Laboratory work No. 5. “Detection of lipids in biological objects”

Goal of the work: prove the presence of lipids in biological objects.

Equipment: rack with test tubes, water bath, pipette, glass cups, sticks, gauze; chicken egg yolk, 1% cholesterol solution in chloroform; concentrated sulfuric acid, acetone.

Progress

1. Detection of lecithin.

Lecithin belongs to the group of phospholipids, is part of cell membranes, and makes up the bulk of brain tissue. Lecithin is insoluble in water and acetone, but soluble in ethyl alcohol, ether and chloroform.

Place part of the yolk of a chicken egg in a glass. While stirring with a stick, add hot ethyl alcohol drop by drop (at the rate of 80 ml per whole yolk). Heat alcohol only in a water bath! When the solution has cooled, filter it into a dry flask. The filtrate should be clear. It must be used immediately.

Add 10 drops of acetone to a dry test tube and add an alcohol solution of lecithin drop by drop. A white precipitate forms.

2. Cholesterol detection.

Cholesterol is a fat-like substance that is of great importance for the body. It is found in the membranes of many organs and tissues and is a precursor of bile acids, vitamin D, sex hormones, and adrenal hormones. The Salkovsky reaction is based on the fact that under the influence of concentrated sulfuric acid, cholesterol is dehydrated to form red-colored cholesterol.

Add 15–20 drops of 1% chloroform solution of cholesterol into a dry test tube and (carefully) add 0.5 ml of concentrated sulfuric acid along the wall of the vessel. A red ring appears at the liquid interface.

Laboratory work No. 6. “Isolation of deoxynucleoprotein from spleen (liver) tissue. Qualitative reaction to DNA"

Goal of the work: prove that nucleic acids are contained in large quantities in the form of compounds with proteins (deoxynucleoproteins - DNP) in tissues rich in nuclei (spleen, thymus, liver, etc.).

Equipment: stand with test tubes, mortar and pestle, glass powder or washed sand, crystallizer, 50 ml and 300 ml graduated cylinders, 1 ml pipettes, notched wooden sticks, water bath, gauze for filtering; 5% sodium chloride solution (containing 0.04% tribasic phosphate), 0.4% sodium hydroxide solution; diphenylamine reagent; spleen (liver) fresh or frozen; Yeast RNA, freshly prepared 0.1% solution.

Progress

1. Isolation of deoxynucleoprotein (DNP) from spleen (liver) tissue.

The method is based on the ability of DNP to dissolve in salt solutions of high ionic strength and precipitate when their concentration decreases.

In a mortar with a small amount of glass powder, thoroughly grind 2–3 g of tissue, gradually adding sodium chloride solution in portions of 10–15 ml (a total of about 40 ml of solution is consumed) for 12–15 minutes.

Filter the resulting viscous solution through two layers of gauze into a crystallizer. Using a cylinder, measure six times (relative to the filtrate) the volume of distilled water and, slowly stirring with a wooden stick, pour into the filtrate. The resulting DNP threads are wound onto a stick, after which they can be transferred to a test tube for further use.

2. Qualitative reaction to DNA.

The method is based on the ability of deoxyribose, which is part of the DNA of deoxyribonucleoproteins, to form blue compounds with diphenylamine when heated in a medium that contains a mixture of glacial acetic and concentrated sulfuric acids. With ribose RNA, a similar reagent gives a green color.

Preparation of diphenylamine reagent: Dissolve 1 g of diphenylamine in 100 ml of glacial acetic acid, add 2.75 ml of concentrated sulfuric acid to the solution.

Add 1 ml of 0.4% sodium hydroxide solution to 1/4 of the DNP sediment (until dissolved). Add 0.5 ml of diphenylamine reagent. Mix the contents of the test tube and place in a boiling water bath for 15–20 minutes. Note the characteristic coloration.

Laboratory work No. 7. “Structural features of prokaryotic and eukaryotic cells”

Goal of the work: based on the study of cells of bacteria (prokaryotes), plants and animals (eukaryotes), to discover the main similarities in the structure of bacteria, animals and plants as an indicator of the unity of organization of living forms.

Equipment: microscope; slides and coverslips; pipettes, glasses of water, tweezers, scalpels, iodine solution, aqueous solution of ink; fuchsin, methylene blue solution, pieces of meat, fish or egg white, onion; table of the structure of bacterial, plant and animal cells.

Progress

1. Prepare infusions in advance from various products: meat, fish, egg whites.

Grind a small amount of material and place it in a flask, add chalk to the tip of a scalpel. Fill 2/3 of the volume with water. Keep the flask with the infusion in a warm place (in a dark place) for 3–5 days. During this time, many different bacteria accumulate in the environment.

2. Place a drop of infusion on a glass slide. Examine the drug using a ×40 lens, but you can also try a ×90 lens (a temporary drug is prepared according to the rules described in the previous work).

3. Add a drop of mascara. Against the general background, bacterial cells are unstained.

4. Draw bacterial cells.

5. Prepare a temporary plant cell preparation. Nuclei in unstained cells are not visible.

Separate the fleshy scale from a piece of onion. There is a thin film on the inside that needs to be removed and cut off. Place a piece of film on a glass slide, pipette the iodine solution, drop it onto the film, and cover with a coverslip.

You can prepare a preparation of an elodea leaf, in which chloroplasts are visible - green plastids.

6. Examine the preparation at low magnification. Large round nuclei in the cells are stained yellow with iodine.

7. Turn the microscope to high magnification and find the cell membrane. In the nucleus you can see 1–2 nucleoli, sometimes the granular structure of the cytoplasm is visible. Unstained voids in the cytoplasm of cells are vacuoles.

8. Draw several cells. Label: membrane, cytoplasm, nucleus, vacuoles (if visible).

9. On the finished preparation, examine the animal cells and sketch them. The figure should indicate: membrane, cytoplasm, nucleus.

10. Have a joint discussion. What provisions of the cell theory can be confirmed by the results of the work performed?

Laboratory work No. 8. “Physiological properties of the cell membrane”

Goal of the work: show that the cell membrane has selective permeability, demonstrate the role of the membrane in the process of phagocytosis and pinocytosis, and also become familiar with cell plasmolysis - the process of separation of the protoplast (cell contents) from the cell walls.

Equipment: microscopes, cover glasses and slides, scalpels, dissecting needles, filter paper, pipettes, ink; culture of ciliates or amoebas, tissue culture on a nutrient medium, pieces of elodea leaves; solutions: potassium chloride, calcium chloride, magnesium chloride,
2% albumin solution, 10% sodium chloride solution; distilled water.

Progress

1. Place ciliates, amoebae or pieces of cultured tissue in a 10% solution of sodium or potassium chloride. Prepare a preparation for the microscope. Cell shrinkage can be seen, which indicates permeability of the cell membrane. In this case, water leaves the cell into the environment.

2. Transfer the cells to a drop of distilled water or remove the solution from under the cover glass using filter paper and replace it with distilled water. Observe how the cells swell, because... water flows into them.

3. Place ciliates or pieces of cultured tissue in a low concentration solution of calcium chloride or magnesium chloride. Ciliates and cultured cells continue to live. Calcium and magnesium ions reduce the permeability of the cell membrane. There is no movement of water through the shell.

4. Place the amoebas in a drop of 2% albumin solution (chicken egg white). Prepare a preparation for the microscope. After some time, vesicles, protrusions, and tubules form on the surface of the amoebas. It seems that the surface of the amoebas is “boiling.” This is accompanied by intense movement of liquid near the surface of the membrane. Drops of liquid are surrounded by protrusions of cytoplasm, which then close together. Pinocytotic vesicles sometimes appear suddenly. This suggests that liquid droplets along with the substances dissolved in it are quickly captured. Pinocytosis is caused by substances that reduce the surface tension of the cell membrane. For example, amino acids, some salts.

Inject a little ink into the drop of liquid in which the amoebas are located. Prepare the drug. After some time, the amoebas begin to slowly move towards the grains of the carcass, releasing pseudopodia (falsepods). Grains of carcass attach to the surface of the pseudopodia, are surrounded by them, and after some time find themselves immersed in the cytoplasm. Under a microscope, the phenomenon of phagocytosis in amoeba is observed.

Literature for teachers

1. Welsh U., Storch F. Introduction to cytology. Translation with him. – M.: Mir, 1986.
2. Zavarzin A.A. and etc. Cell biology. – St. Petersburg: St. Petersburg State University Publishing House, 1992.
3. Swanson K., Webster P.– M.: Mir, 1982.
4. Lamb M. Biology of aging. – M.: Mir, 1980.
5. Markosyan A.A. Physiology. – M.: Medicine, 1968.
6. Liberman E.A.
7. Ermolaev M.V. Biological chemistry. – M.: Medicine, 1984.
8.Ruvinsky A.O. General biology. – M.: Education, 1993.

Literature for students

1. Green N., Stout W., Taylor D. Biology. In 3 volumes - M.: Mir, 1993.
2. De Duve K. Journey into the world of a living cell. – M.: Mir, 1982.
3. Liberman E.A. Living cell. – M.: Mir, 1987.
4. Kemp P., Arms K. Introduction to biology. – M.: Mir, 1988.

BELARUSIAN STATE UNIVERSITY

DEPARTMENT OF BIOLOGY

Department of Plant Physiology and Biochemistry

PHYSIOLOGY

PLANT

CELLS

for laboratory workshops

"Plant Physiology"

for students of the Faculty of Biology

V. M. Yurin, A. P. Kudryashov, T. I. Ditchenko, O. V. Molchan, I. Smolich Recommended by the Academic Council of the Faculty of Biology June 16, 2009, protocol No. Reviewer Candidate of Biological Sciences, Associate Professor M. A Juice Physiology of plant cells: method. recommendations for laboratory classes of the workshop “Plant Physiology” for F students of the Faculty of Biology / V. M. Yurin [et al.].

– Minsk: BSU, 2009. – 28 p.

This manual is an integral element of the educational and methodological complex in the discipline “Plant Physiology” and includes laboratory work in the section “Physiology of Plant Cells”.

Intended for students of the Faculty of Biology studying in the specialties “Biology” and “Bioecology”.

UDC 581. BBK 28. © BSU,

FROM THE AUTHORS

Methodological recommendations for laboratory classes are an integral part of the “Plant Physiology” course. The purpose of the publication is to enhance the independent work of students, taking into account the fact that the individual learning process must be effective. The workshop on the course “Plant Physiology” is intended to consolidate theoretical material, acquire practical work skills and familiarize yourself with the basic methods of researching the physiological processes of plants. Students are offered assignments that detail the factual material that they must master on their own.

This will allow you to use classroom time more efficiently.

1. PLANT CELL HOW

OSMOTIC SYSTEM

Osmotic systems are systems consisting of two solutions of substances of different concentrations, or a solution and a solvent, separated by a semi-permeable membrane. An ideal semi-permeable membrane allows solvent molecules to pass through but is not permeable to solute molecules. In all biological systems, water is the solvent. The difference in the composition and concentration of substances on both sides of a semi-permeable membrane is the cause of osmosis - the directed diffusion of water molecules through a semi-permeable membrane.

If we abstract from the detailed structure of the plant cell and consider it from the point of view of the osmotic model, then it can be argued that the plant cell is a living osmotic system.

The plasma membrane is semipermeable, and the cytoplasm and tonoplast act as a single unit. Outside the semipermeable membrane is a cell wall that is highly permeable to water and substances dissolved in it and does not interfere with the movement of water. The main role of the osmotic space of the cell is played by the vacuole, which is filled with an aqueous solution of various osmotically active substances - sugars, organic acids, salts, water-soluble pigments (anthocyanins, etc.). However, this is a rather simplified idea of ​​a cell as an osmotic system, since any cytoplasmic organelle surrounded by a membrane is also an osmotic cell. As a result, osmotic movement of water occurs between the individual organelle and the cytosol.

PLANT CELL MODELS

Introductory remarks. The unique physicochemical characteristics of biomembranes ensure the flow of water and the creation of high hydrostatic pressure (turgor) in the plant cell, the preservation of an anisotropic distribution of substances between the cell and its environment, the selective absorption and release of substances, and a number of other functions.

The hypothesis about the existence of a plasma membrane on the cell surface was put forward in the second half of the 19th century. The scientific justification for this hypothesis (concept) was given by W. Pfeffer based on an explanation of the phenomena of plasmolysis and deplasmolysis. According to Pfeffer, this membrane had the property of “semi-permeable”, that is, it was permeable to water and impermeable to substances dissolved in water. In subsequent years, studies were carried out that made it possible not only to prove the existence of such a structure on the surface of the cell, but also to study some of the properties of this structure invisible in optical microscopes. However, until the second half of the twentieth century. biomembranes remained only hypothetical structures of a living cell. Therefore, to demonstrate certain properties of the plasma membrane and explain the patterns of functioning of the mechanisms associated with the plasma membrane, researchers created cell models (“artificial cells”).

At different periods of time, model systems appeared - “artificial cells” by Pfeffer, Traube, Jacobs and others. The first two of the mentioned models demonstrated the phenomena of osmosis, the third - the patterns of transfer of weak electrolytes through the biomembrane. When performing laboratory work, it is proposed to create “artificial cell” model systems according to Traube and Jacobs (modified).

When forming the “artificial cell” models of Pfeffer and Traube, at the interface between solutions of yellow blood salt and copper sulfate, a water-insoluble amorphous mass of iron sulfate copper is formed, which has almost ideal osmotic properties - permeability to water and impermeability to dissolved substances. Since an iron-copper membrane separates two solutions, the direction and magnitude of water flow through it will be determined by the difference in the chemical potentials of water molecules on opposite sides of the membrane. If such a membrane separated two solutions of the same substance, then the chemical potential of the water molecules would be higher in the more dilute solution, and water would move towards the solution of lower concentration. When determining the direction of water movement in a system containing different substances on both sides of the membrane, the degree of dissociation of substances, valence and permeability of the membrane for ions should be taken into account. To simplify the discussion of the experiment to obtain an “artificial cell” according to Traube, we assume that the membrane made of iron sulfide copper is absolutely impermeable to dissolved substances, the degree of dissociation of yellow blood salt and copper sulfate in solutions is the same. In this case, to compare the values ​​of the chemical potential of water molecules, you can use the normal concentrations of the indicated salts.

The basic principles of the process of diffusion of substances of different polarities through plasma membranes were established in the first half of the twentieth century. According to research by Collander and Barlund, the permeability coefficient of a membrane to any substance can be predicted by the molecular weight of the latter and its equilibrium distribution coefficient (kр) between water and vegetable oil:

where CM and SV are the concentrations of the substance that are established in a system of solvents in contact with each other - oil and water - in a state of equilibrium. For most substances diffusing through the plasma membrane, there is a direct proportionality between the product Pi M i and kр (Pi is the permeability coefficient of the membrane with respect to substance i; Mi is the molecular weight of substance i).

The kр coefficient in this case acts as a quantitative measure of the degree of hydrophobicity: more hydrophobic substances accumulate in oil and are characterized by a large kр value, while hydrophilic substances, on the contrary, accumulate in the aqueous phase, for which the kр value is smaller. In accordance with this, non-polar compounds should penetrate into the cell as a result of the process of diffusion through the layer of membrane lipids more easily than polar ones. The degree of hydrophobicity is determined by the structure of the molecule of the substance. However, the hydrophobicity of a substance largely depends on the degree of ionization of its molecules in solution. In turn, the degree of ionization of many organic and inorganic substances (weak electrolytes) is determined by the pH value of the solution.

Jacobs's "artificial cell" models the selective permeability of the plasma membrane of plant cells towards electrically neutral molecules of weak electrolytes. In his original design of an “artificial cell,” Jacobs used a flap of frog skin as an analogue of the plasmalemma. In the proposed work, a film made of a hydrophobic (polymer) material is used as a model of the plasmalemma. This was done not only for reasons of humanity - the polymer film more clearly models the physicochemical properties of the lipid bilayer of the plasmalemma.

Being a weak base, ammonium exists in aqueous solutions in the form of NH3 and NH4+, the concentration ratio of which depends on the pH of the medium and for dilute aqueous solutions is determined by the dissociation constant pKa, which at 25 °C is equal to 9.25:

where and are the concentrations of ammonia molecules and ammonium ions, respectively.

If only uncharged ammonia molecules can penetrate the membrane, then it is easy to show that the concentrations of ammonium ions on different sides of the membrane in equilibrium will depend on the pH of the solutions in contact with the membrane. To demonstrate the process of ammonia transport across a membrane in Jacobs's "artificial cell", its ability to shift pH is used.

Goal of the work. Obtain “artificial cells” using the Traube and Jacobs methods and observe the phenomenon of osmosis - the movement of water through a semi-permeable membrane along a gradient of osmotic potential.

Materials and equipment: 1.0 N solutions of yellow blood salt, copper sulfate, ammonium chloride, sodium hydroxide and hydrochloric acid, 1% aqueous alcohol solution of neutral red, universal indicator paper, fragments of glass tubes melted at the end, polymer film, threads, test tubes , 3 glasses with a capacity of 150–200 ml, stopwatch.

1. Preparation of Traube’s “artificial cell”. By dilution, prepare 1.0 N solution of yellow blood salt (K4Fe(CN)6), 0.5 N and 1.N solutions of copper sulfate (CuSO45 H2O). Take two test tubes. Pour 0.5 N into one, and 1.0 N solution of copper sulfate into the other. Carefully pipet along the wall of the test tubes into each 1.0 N solution of yellow blood salt. On the contact surface of solutions of copper sulfate and yellow blood salt, a membrane of iron sulfide copper is formed:

An amorphous precipitate of iron-synoxide copper has almost ideal osmotic properties, therefore, when the chemical potential of H2O molecules differs, a flow of water should be observed, which leads to a change in the volume of the “artificial cell”. It should be noted that the membrane made of iron sulfide copper has weak elasticity. Therefore, when the volume of the “artificial cell” increases, the membrane breaks.

Exercise. Monitor the behavior of “artificial cells” in 0.5 N and 1.0 N solutions of copper sulfate. Sketch "artificial cells"

and describe the dynamics of changes in their shape.

2. Obtaining an “artificial Jacobs cell”. By dilution, prepare 200 ml of 0.5 N ammonium chloride solution and 100 ml of 0.5 N sodium hydroxide. Pour the sodium hydroxide solution into a glass, and divide the ammonium chloride solution into two equal parts and pour them into glasses with a capacity of 150–200 ml. Using indicator paper and 1.0 N solutions of hydrochloric acid and sodium hydroxide, adjust the acidity of the solution in the first glass to pH 9.0, and in the second to pH 7.0.

Take 3 glass tube fragments. Place a piece of polymer film on the melted end of each and carefully tie them with thread. Add 5-10 drops of neutral red solution to 50 ml of water and slightly acidify the medium with 1-2 drops of hydrochloric acid.

Fill the “artificial Jacobs cells” (fragments of glass tubes with membranes) with the indicated indicator solution. Place the “artificial Jacobs cells” in beakers containing solutions of sodium hydroxide and ammonium chloride so that these media are in contact with the polymer membrane.

Ammonia is able to diffuse through the hydrophobic phase of the polymer membrane. And since its concentration inside the “artificial cell” is negligible, NH3 molecules are transferred from the solution into the “cell” and cause alkalization of the contents of the glass tube, which is noted by the disappearance of the crimson-red color of the “intracellular” contents.

Exercise. Determine the time required for the red color of the indicator to disappear in each variant of the experiment.

1. Why does the salt concentration increase near the surface of the “artificial cell” in a 0.5 N solution of copper sulfate?

2. Why does an “artificial cell” swell in a 0.5 N solution of copper sulfate, but in a 1.0 N solution its surface is stable?

3. On what factors does the degree of dissociation of weak acids and bases depend?

4. Why, when placing an “artificial cell” in a solution of sodium hydroxide, does the neutral red color disappear?

5. Why, when placing an “artificial cell” in a neutral solution of ammonium chloride, is there a shift in the pH of the “intracellular” contents to weakly basic values?

6. What is osmosis?

7. What solutions are called hypo-, iso- and hypertonic?

PHENOMENON OF PLASMOLYSIS AND DEPLASMOLYSIS

PLANT CELL

Introductory remarks. The process of water leaving a plant cell and entering the cell through a semi-permeable membrane can be traced by observing the phenomena of plasmolysis and deplasmolysis. When a cell is placed in a solution that is hypertonic in relation to the cell sap, plasmolysis occurs - the separation of the protoplast from the cell wall due to a decrease in its volume due to the release of water from the cell into the external solution. During plasmolysis, the shape of the protoplast changes. Initially, the protoplast lags behind the cell wall only in some places, most often in the corners. Plasmolysis of this form is called angular. With increasing duration of incubation of a plant cell in a hypertonic solution, the following form of plasmolysis is observed - concave plasmolysis. It is characterized by the preservation of contacts between the protoplast and the cell wall in separate places, between which the separated surfaces of the protoplast acquire a concave shape. Gradually, the protoplast breaks away from the cell walls over the entire surface and takes on a rounded shape. This type of plasmolysis is called convex plasmolysis.

After replacing the external solution with clean water, the latter begins to flow into the cell. The volume of the protoplast increases and deplasmolysis occurs. After its completion, the protoplast again fills the entire volume of the cell.

Goal of the work. Prove, based on the phenomena of plasmolysis and deplasmolysis, that a plant cell is an osmotic system.

Materials and equipment: microscope, slides and cover glasses, safety razor blade, dissecting needle, tweezers, 1 M sucrose solution, filter paper, onion bulb.

From the convex side of the surface of the onion scales, the cells of which are colored purple due to the presence of anthocyanins in the vacuoles, the epidermis is removed with a dissecting needle, placed in a drop of water on a glass slide, covered with a coverslip and examined under a microscope. Then replace the water with a 1 M sucrose solution. To do this, apply a large drop of solution to a glass slide next to the coverslip and suck out the water with a piece of filter paper, placing it on the other side of the coverslip. Repeat this technique 2-3 times until the water is completely replaced with the solution. The preparation is examined under a microscope. A gradual lag of the protoplast from the cell walls is detected, first in the corners, and then along the entire surface of the walls. Eventually, the protoplast is completely separated from the cell wall and takes on a rounded shape.

Then, using the method described above, replace the 1 M sucrose solution with water. Water enters the cell, which leads to an increase in the volume of the protoplast, which gradually takes its previous position. The cell returns to its original state.

Exercise. Draw the observed forms of plasmolysis, as well as the stages of deplasmolysis. Formulate conclusions.

1. What structural features of a plant cell give it the properties of an osmotic system?

2. What is plasmolysis? Describe the main forms of plasmolysis.

3. What is deplasmolysis? Under what conditions is it observed?

DETERMINATION OF OSMOTIC PRESSURE

CELL JUICE PLASMOLYTIC

BY METHOD

Introductory remarks. When two solutions containing different amounts of dissolved substances come into contact, due to the inherent thermal motion of molecules, mutual diffusion occurs, which leads to equalization of the concentration of dissolved substances throughout the volume, which is equivalent to the situation of mixing liquids. If these solutions are separated by a semi-permeable membrane that retains molecules of dissolved substances, then only solvent (water) molecules will pass through the contact boundary of the solutions. Moreover, a unidirectional flow of water through the membrane (osmosis) occurs. The pressure that must be applied to one of the solutions of the system in order to prevent the solvent from entering it is called osmotic pressure. The osmotic pressure of a solution is directly proportional to its concentration and absolute temperature. Van't Hoff established that the osmotic pressure of dilute solutions obeys gas laws and can be calculated using the formula:

where R is the gas constant (0.0821); Т – absolute temperature (273 оС + t оС) of the solution; C is the concentration of the dissolved substance in moles; i – isotonic coefficient.

The value of the isotonic coefficient is determined by the characteristics of the dissolution processes of the substance. For non-electrolytes (for example, for sucrose) i is equal to 1. For electrolyte solutions, the value of i depends on the number of ions into which the molecule breaks up and on the degree of dissociation. The values ​​of i for NaCl solutions are given in the table.

Values ​​of the isotonic coefficient of sodium chloride solutions Concentration of NaCl Value i The value of the osmotic pressure of cell sap expresses the ability of a plant cell to “absorb” water and indicates the possibility of plant growth on soils of different water-holding strength. At the same time, an increase in the osmotic pressure of cell sap during drought is a criterion for plant dehydration and the need for watering.

The plasmolytic method for determining the osmotic pressure of cellular contents is based on the fact that the osmotic pressure of solutions, which determines the movement of water through the membrane, can be created by various substances (osmolytics). Therefore, to determine the osmotic pressure of cell sap, knowledge of its qualitative composition and concentration of individual substances is not required, but one should find the concentration of a substance in the external solution at which there will be no movement of water through the plasmalemma in the absence of turgor and plasmolysis. To do this, sections of the tissue under study are immersed in a series of solutions of known concentrations, and then they are examined under a microscope. Based on the fact that only hypertonic solutions can cause plasmolysis, the weakest of them is found, in which only initial plasmolysis is detected in individual cells. The following more dilute solution will not plasmolyze the cells.

Consequently, the concentration of an isotonic solution for these cells will be equal (with a certain degree of error) to the arithmetic mean between the concentrations of neighboring solutions.

For convenience, the work is carried out with tissues whose cells contain anthocyanins in the cell sap: the epidermis of blue onion scales, the lower epidermis of a tradescantia leaf. Solutions of sucrose or NaCl are used as a plasmolytic.

Materials and equipment: microscope, slides and coverslips, safety razor blade, dissecting needle, solutions of 1 M NaCl and 1 M sucrose, tradescantia leaves or blue onion bulbs.

Using 1 M sucrose or NaCl solution, prepare by diluting 5 ml solutions according to the table.

After thoroughly mixing the solutions, pour them into glass bottles or crucibles, where you place 2-3 sections of the tissue being tested for 30 minutes.

In this case, it is necessary to ensure that the sections do not float on the surface, but are immersed in liquid (if the section floats, it should be “drowned” using a dissecting needle). Cover the bottles with lids or glass slides to prevent evaporation.

After the specified incubation time has passed, examine the sections under a microscope in a drop of the appropriate solution (not in water!) in the same sequence in which they were immersed in the solutions. The glass rod or pipette with which the solution was applied to the glass slides must be thoroughly rinsed with distilled water after each solution and wiped with a napkin or filter paper.

Exercise. Determine the presence of plasmolysis and its degree in the tissue under study. The degree of plasmolysis is expressed by the concepts: “strong”, “weak”, “initial”, “lack of plasmolysis”. Enter the results into the table.

Degree of plasmolysis Isotonic concentration, M Osmotic pressure of cell sap in atm and kPa Establish the isotonic concentration of sodium chloride, i.e., the NaCl content that creates an osmotic pressure similar to cell sap in the tissue under study. Calculate the osmotic pressure using equation (1). Using the coefficient 101.3, calculate the osmotic pressure in kPa.

1. What is osmotic pressure?

2. How is the osmotic pressure calculated?

3. What does the value of the isotonic coefficient depend on?

4. The criterion for which process is an increase in the osmotic pressure of cell sap?

2. PROPERTIES OF CELL MEMBRANES

The most important property of cell membranes is selective permeability. The outer cytoplasmic membrane, separating the cell from the environment, controls the transport of substances between the cell and free space. Intracellular membranes, due to their inherent selective permeability, provide a compartmentalization function that allows the cell and organelles to retain the necessary enzymes and metabolites in small volumes, create a heterogeneous physicochemical microenvironment, and carry out various, sometimes oppositely directed, biochemical reactions on different sides of the membrane.

The permeability of cell membranes to various substances can be a criterion for cell viability. Selective permeability of the membrane is maintained as long as the cell remains alive.

STUDYING SELECTIVE PERMEABILITY

PLANT CELL PLASMALEMMAS

Introductory remarks. The permeability of the plasma membrane for various substances can be compared on the basis of simple observations characterizing the duration of plasmolysis in plant cells located in hypertonic solutions of the substances under study. In the case of a sufficiently low permeability of the plasmalemma for a dissolved substance or a complete absence of the ability of its molecules to freely diffuse into the plant cell, persistent plasmolysis will occur, in which plasmolyzed cells can remain in an unchanged state for a long time. However, if the molecules of the dissolved substance pass through the membrane, but more slowly than the water molecules, then the plasmolysis that begins is temporary and soon disappears. As a result of the gradual penetration of the solute into the cell, the flow of water from the external solution along the concentration gradient will be observed, which will ultimately cause the cell to transition to a deplasmolyzed state.

Goal of the work. Compare the permeability of cell membranes to various substances based on the observation of persistent and temporary plasmolysis.

Materials and equipment: microscope, slides and cover glasses, safety razor blade, dissecting needle, tweezers, 1 M sucrose solution, 1 M urea solution, 1 M glycerin solution, filter paper, onion bulb.

A drop of solution is applied to three object steles: on one - a 1 M sucrose solution, on the other - a 1 M solution of urea, on the third - a 1 M solution of glycerol. A fragment of the colored onion epidermis is placed in each drop, covered with coverslips and examined under a microscope. Find areas in which plasmolyzed cells are clearly visible. The time of the beginning of plasmolysis is noted - the beginning of observation. Leave the preparations for 10–30 minutes, then examine them again under a microscope. In a solution of sucrose, persistent plasmolysis is observed, and in solutions of urea and glycerol - temporary. The reason for deplasmolysis in the last two solutions is the permeability of the plasma membrane for urea and glycerol molecules.

Exercise. Conduct a study of the characteristics of plasmolysis of plant cells in solutions of various substances. Record the observation results in the table, noting the degree of plasmolysis every 10 minutes after the start of observations. Based on the analysis of the experimental results, identify differences in the duration of preservation of the plasmolyzed state caused by various osmolytics, and draw a conclusion about the relative permeability of the plasmalemma for the substances under study.

Solute Note: +++ – strong plasmolysis, ++ – moderate plasmolysis, + – weak plasmolysis.

1. What is selective permeability of cell membranes?

2. Which substances penetrate cell membranes more easily?

3. How can the property of selective permeability be used to determine the viability of a plant cell?

STUDYING THE DIFFUSION OF NEUTRAL

RED THROUGH PLASMALEMMA

PLANT CELL

Introductory remarks. The plasma membrane isolates intracellular contents from the external environment. The exchange of substances between the intracellular contents and the environment surrounding the cell occurs through their transport through the membrane. The lipid bilayer is a barrier to the movement of substances. Most exogenous physiologically significant substances enter the cell as a result of the functioning of passive and active transport systems on the plasmalemma. However, simple passive diffusion through the lipid bilayer, which is a hydrophobic phase, is also possible.

The basic patterns of diffusion of substances through the lipid bilayer were established at the end of the 19th and beginning of the 20th centuries, i.e., during that period of time when biomembranes remained only hypothetical structures of the cell. It is the fact that hydrophobic substances penetrate inside the cell better than hydrophilic ones that was the basis for the researchers’ assumption about the presence of lipids in the membrane.

The process of diffusion of substances through a membrane obeys Fick's first law, the mathematical expression of which, as applied to a membrane, is described by the formula:

where Pi is the membrane permeability coefficient for substance i; CiII and CiI are the concentrations of substance i on both sides of the membrane.

Weak acids and bases are characterized by the fact that the degree of ionization of their molecules in dilute solutions depends on pH (see Laboratory work 1, formula (2)). This means that the degree of dissociation of weak electrolyte molecules in the range of pH values ​​numerically equal to pKa is 50%. When pH decreases by one, more than 90% of the weak base molecules will be ionized, and when pH increases by the same amount, less than 10% will be ionized.

Back in the first half of the twentieth century, it was demonstrated that electrically neutral, non-ionized molecules of weak electrolytes penetrate quite well through the plasma membrane into plant cells, while the membrane turns out to be practically impenetrable for the corresponding ions. For example, the permeability coefficients of the plasmalemma for ammonia and ammonium ion differ by more than 100-fold. Thus, the pH values ​​shift by only 1–2 units. leads to a more than 10-fold change in the concentration of the forms of substance molecules transported through the membrane.

Among weak electrolytes, acid-base indicators are of particular interest, since the molecules of these substances are characterized by a change in their optical properties upon ionization. In addition, due to the characteristic color of solutions of these compounds, it is quite easy to determine their content colorimetrically. Neutral red (NR) is a weak base. Ionized NA molecules (at pH 6.8 and below) color solutions an intense crimson color. As pH increases from 6.8 to 8.0, a gradual change in color to pale yellow occurs due to a decrease in the degree of dissociation of NA molecules. In alkaline solutions, electrically uninfected NA molecules, which are well transported through the lipid bilayer of the plasma membrane, predominate, and in acidic solutions, NA ions, which are weakly permeable to the membrane, predominate.

NA molecules entering the cell through the plasmalemma can diffuse through other cell membranes, however, having penetrated inside the vacuole (the acidic compartment of a plant cell), NA molecules are ionized, coloring the contents of the vacuole crimson. In this case, NK ions turn out to be “closed” in the space of the vacuole, i.e., they tend to accumulate.

Goal of the work. To study the patterns of diffusion of neutral red through the plasmalemma of a plant cell. Materials and equipment: scissors, aqueous-alcoholic solution of neutral red, decinormal solutions of sodium hydroxide and hydrochloric acid, universal indicator paper, Petri dishes, microscope, stopwatch, Nitella flexilis algae culture.

Add 5 drops of neutral red solution to 100 ml of water.

Pour this solution equally into 4 Petri dishes. Controlling the acidity of the contents of the Petri dishes with universal indicator paper using HCl and NaOH solutions, bring the acidity in the first Petri dish to pH 9.0, in the second to pH 8.0, in the third to pH 7.0, in the fourth to pH 5.0. Label the Petri dishes.

Using scissors, carefully remove 8–12 algae internodes from the Nitella flexilis thallus. Examining the internodes under a microscope, make sure that the prepared cells are native: living, undamaged cells retain continuous rows of chloroplasts located parallel to the light line; in addition, intense movement of the cytoplasm is observed - cyclosis.

Place 2-3 cells of algae internodes into Petri dishes.

Start the stopwatch.

Exercise. Determine the time required to stain the algae cells in each experiment. To do this, after 5 minutes, compare the cells of the internodes of the algae of each of the variants by color intensity. Repeat the operation after 10, 20, 30 minutes. Record the observation results in the table. Draw conclusions regarding weak base forms diffusing through the membrane.

pH value of the medium Note: +++ – intense color, ++ – medium color, + – weak color, – no color.

1. On what factors does the degree of dissociation of weak acids and bases depend?

2. Why are biomembranes more permeable to undissociated forms of weak electrolytes?

3. Under what conditions is the accumulation of a weak electrolyte in the cell observed?

CHANGE IN THE PERMEABILITY OF TONOPLAST

AND PLASMALEMMAS FOR BETACYANINE UNDER

ACTION OF PHYSICAL AND CHEMICAL

FACTORS

Introductory remarks. The selective permeability of cell membranes changes under the influence of various factors. The influence of any substances or conditions on membrane permeability can be determined by measuring the release of various metabolites from the cell.

Betacyanin, the beet pigment, is a relatively large, highly water-soluble molecule found in cell sap.

To enter the external environment, the betacyanin molecule must pass through the tonoplast, the main cytoplasmic matrix and the plasmalemma. Tonoplasts of living cells are impenetrable to molecules of this pigment. Diffusion of betacyanin from the vacuole into the medium can occur quite quickly under the action of various factors or agents that cause an increase in membrane permeability. By measuring the optical density of the incubation medium after a certain period of time, it is possible to assess the degree of influence of a particular factor on the permeability of membranes.

Goal of the work. Determine the effect of temperature, as well as acids and alcohols on the permeability of cell membranes for betacyanin by its release into the external solution.

Materials and equipment: distilled water, 30% acetic acid solution, 50% ethanol solution, filter paper, test tubes, test tube rack, water bath, spectrophotometer or photocolorimeter, beetroot.

After removing the integumentary tissue, the beet root is cut into cubes (cube side 5 mm) and thoroughly washed with water for 5–10 minutes to remove the pigment released from the damaged cells.

Then they are placed one at a time in each of 4 test tubes, into which 5 ml of various media are poured in accordance with the experimental scheme: distilled water (2 test tubes), solutions of acetic acid and ethanol.

The first test tube with distilled water is left in a stand, and the contents of the second are heated in a water bath for 2–3 minutes. After 30 minutes, all test tubes are shaken vigorously, the beet cubes are removed, and the color intensity of the solutions is determined using a photocolorimeter with a green light filter or a spectrophotometer = 535 nm.

Optical density of the solution, Color intensity, Experimental option Assignment. Do your research. Enter the results of optical density measurements into the table. Identify differences in the permeability of the tonoplast and plasmalemma to betacyanin in beet root cells exposed to various factors, and draw a conclusion about the reasons for these differences.

1. What is the significance of selective permeability of cell membranes?

2. What does the selective permeability of plant cell membranes depend on?

3. PROPERTIES OF CYTOPLASMA

The main volume of cytoplasm that fills the space between cellular organelles is called cytosol. The proportion of water in the cytosol is approximately 90%. Almost all the main biomolecules are contained in dissolved form in the cytosol. True solutions form ions and small molecules (alkali and alkaline earth metal salts, sugars, amino acids, fatty acids, nucleotides and dissolved gases). Large molecules, such as proteins, form colloidal solutions. A colloidal solution can be a sol (non-viscous) or a gel (viscous). The intensity of most intracellular processes depends on the viscosity of the cytosol.

The most important property of the cytoplasm is its active movement.

This is a characteristic feature of a living plant cell, an indicator of the activity of its vital processes. The movement of the cytoplasm ensures intracellular and intercellular transport of substances, the movement of organelles within the cell, and plays an important role in irritability reactions. Elements of the cytoskeleton—microfilaments and microtubules—take part in its implementation. The energy source for this movement is ATP. Cytoplasmic movement (cyclosis) is one of the most sensitive indicators of cell viability. Many even minor influences stop or, on the contrary, accelerate it.

INFLUENCE OF POTASSIUM AND CALCIUM IONS ON

VISCOSITY OF PLANT CELL CYTOPLASM

Introductory remarks. Individual cations can significantly change the viscosity of the cytoplasm. It has been established that potassium ions contribute to an increase in its water content and a decrease in viscosity. The lower viscosity of the cytoplasm favors the course of synthetic processes and intracellular transport of substances, but reduces the resistance of plant cells to unfavorable external conditions. Unlike potassium, calcium increases the viscosity of the cytoplasm. With higher cytosol viscosity, physiological processes occur more slowly, which increases the cell's resistance to unfavorable environmental conditions.

Changes in the viscosity of the cytoplasm under the influence of potassium and calcium ions can be judged by the form of plasmolysis in cells in hypertonic solutions of their salts. When plant cells are incubated for a long time in solutions containing potassium ions, cap plasmolysis is observed. In this case, potassium ions pass through the plasmalemma into the cytoplasm, but rather slowly penetrate through the tonoplast into the vacuole. As a result of swelling of the cytoplasm, the protoplast takes on a convex shape, separating only from the transverse sections of the cell walls, from which the formation of so-called “caps” is observed. The increase in cytoplasmic viscosity caused by calcium is easy to detect by observing the change in the shape of the plasmolyzing protoplast: if the plasmolytic contains calcium, then concave plasmolysis often turns into a convulsive form.

Goal of the work. To study the nature of the influence of potassium and calcium ions on the viscosity of the cytoplasm of a plant cell based on observations of cap and convulsive plasmolysis.

Materials and equipment: microscope, slides and cover glasses, safety razor blade, dissecting needle, tweezers, 1 M KNO3 solution, 1 M Ca(NO3)2 solution, filter paper, onion bulb.

A drop of 1 M potassium nitrate solution is placed on one glass slide, and a 1 M calcium nitrate solution is placed on the other. A piece of onion epidermis, removed from the concave surface of the same onion scale, is placed in both drops and covered with coverslips. After 30 minutes, the preparations are examined under a microscope in the solutions in which they were located. The phenomenon of plasmolysis is observed. In some epidermal cells kept in a KNO3 solution, the cytoplasm forms “caps” on the side of the transverse cell walls, the appearance of which is due to an increase in the hydration of the cytosol under the influence of potassium ions. Calcium ions, on the contrary, increase the viscosity of the cytoplasm, increase its adhesion forces to the cell wall, and the protoplast takes on irregular shapes characteristic of convulsive plasmolysis.

Exercise. Draw the observed forms of plasmolysis. Reveal the dependence of the form of plasmolysis on the viscosity of the cytoplasm in the presence of potassium and calcium ions.

1. How do potassium and calcium ions affect the viscosity of the cytoplasm?

2. Under what conditions is convulsive plasmolysis observed?

3. What causes the formation of “caps” as a result of incubation of cells in a KNO3 solution?

OBSERVATION OF CYTOPLASMA MOVEMENT

PLANT CELLS AND MEASUREMENT OF ITS

SPEED

Introductory remarks. The most convenient for observing the movement of cytoplasm are large plant cells with large vacuoles (cells of internodes of charophyte algae, marine siphon green algae, cells of leaves of aquatic plants Elodea, Vallisneria, etc.). There are several types of cytoplasmic movement. The most common is oscillatory motion. It is considered the least ordered, since in this case some particles are at rest, others slide to the periphery, and others to the center of the cell. The movement is unstable and random. Circulatory movement is characteristic of cells that have cytoplasmic cords crossing the central vacuole. The direction and speed of movement of particles located inside or on the surface of the cytoplasmic layer, as well as in the cytoplasmic layers, are not constant. During rotational movement, the cytoplasm moves only at the periphery of the cell and moves like a drive belt. Movement of this type, in contrast to circulation, has a more or less constant and ordered nature, and therefore is convenient for quantitative study. In addition to the above, there are also cytoplasmic movements, such as gushing and shuttle movements. The types of movement differ from each other conditionally and in the same cell they can move from one to another.

The movement of the cytoplasm can be characterized by determining its speed, which depends not only on the driving force, but also on the viscosity of the cytoplasm. The speed of movement of the cytoplasm can be measured under a microscope by observing the movement of its particles.

Goal of the work. Familiarize yourself with the rotational type of cytoplasmic movement and measure its speed in different plant objects.

Materials and equipment: microscope, slides and coverslips, safety razor blade, dissecting needle, artificial pond water solution, Vallisneria leaf, internodal nitella cells.

A small piece is cut off from the Vallisneria leaf blade with a sharp razor, trying to injure the leaf as little as possible, place it in a drop of water on a glass slide and examine it under a microscope, first at low, then at high magnification. It is not recommended to make sections from the leaf, since the cells are severely injured and movement in them stops. The movement of the cytoplasm can be easily observed by the movement of all chloroplasts in one direction along the cell wall. This movement is called rotational.

To observe cyclosis in Nitella cells, pre-prepared cells are placed in special chambers, which are filled with a solution of artificial pond water. All charophyte algae also exhibit a rotational type of cytoplasmic movement, but the chloroplasts in these cells are immobile. Adjacent directly to the cellulose membrane is a dense and immobile layer of cytoplasm, called ectoplasm. In this layer, chromatophores are fixed, which form one layer of tightly adjacent regular longitudinal rows. Between the vacuole and the ectoplasm layer there is an internal liquid mobile layer of cytoplasm, the so-called endoplasm. Its intensive movement can be observed by the movement of organelles smaller than chloroplasts - small colorless inclusions suspended in the cytoplasm.

To determine the speed of movement of the cytoplasm, use a stopwatch and an eyepiece ruler placed in the eyepiece of the microscope. Using a stopwatch, the time is counted during which a chloroplast or other moving particle passes the distance between two selected divisions of the ocular ruler. Such measurements in the same cell are carried out 3–5 times. To calculate the speed of movement of the cytoplasm, the division value of the ocular ruler is measured. To do this, an object micrometer is placed on the microscope stage, which is examined through an ocular micrometer. Fix the selected lens on the divisions of the object micrometer and count the number of divisions of the object micrometer. The price of ocular micrometer divisions is calculated by the formula where N is the price of ocular micrometer divisions; 10 µm – micrometer division price; b – the number of divisions of the eyepiece micrometer that fit into (a) divisions of the object micrometer.

The speed of particle movement is the ratio of the distance in micrometers to the number of seconds during which a moving particle travels this distance (μm/s).

Exercise. Determine the speed of cytoplasmic movement in the cells of aquatic plants. Enter the measurement results in the table. Make schematic drawings of the cells of the objects considered and use arrows to indicate the direction of movement of the cytoplasm, compare the nature and speed of cyclosis.

Object Type of moving Distance Particle travel time, s Cyclosis speed, 1. What is cytosol?

2. How does the form of plasmolysis depend on the viscosity of the cytoplasm of plant cells?

3. What is the biological significance of the movement of the cytoplasm?

4. What are the main types of cytoplasmic movement?

5. What determines the speed of cytoplasmic movement?

From the authors……………………………………………………….

1. PLANT CELL AS OSMOTIC

SYSTEM………………………………………………………….

Laboratory work Models of plant cells……………………………………... Laboratory work The phenomenon of plasmolysis and deplasmolysis of a plant cell..……. Laboratory work Determination of the osmotic pressure of cell sap by the plasmolytic method…………………………………….……………. 2. PROPERTIES OF CELL MEMBRANES………..…………..

Laboratory work Study of the selective permeability of the plasmalemma of a plant cell…………………….…………………………….. Laboratory work Study of the diffusion of neutral red through the plasmalemma Laboratory work Changes in the permeability of the tonoplast and plasmalemma for betacyanin under the influence physical and chemical factors... 3. PROPERTIES OF CYTOPLASMA……………………………... Laboratory work The influence of potassium and calcium ions on the viscosity of the cytoplasm of a plant cell…………………………………… ..……………………. Laboratory work Observing the movement of the cytoplasm of plant cells and measuring its speed……………………………………………………….

PLANT CELL PHYSIOLOGY

Workshop "Plant Physiology"

for students of the Faculty of Biology Responsible for the issue A.P. Kudryashov Signed for publication on August 31, 2009. Format 6084/16. Offset paper.

Times typeface. Conditional oven l. 1.63. Academic ed. l. 1.62. Circulation 50 copies. Zach.

Belarusian State University 220030, Minsk, Independence Avenue, 4.

Printed from the customer's original layout using copying equipment of the Belarusian State University.

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SECTION 2

LABORATORY EXERCISES

Laboratory work No. 1

Comparison of membrane permeability of living and dead cells

Exercise: identify differences in membrane permeability of living and dead cells and draw conclusions about the reasons for these differences.

Materials and equipment: test tubes, test tube rack, scalpel, alcohol lamp or gas burner, 30% acetic acid solution, beetroot.

Operating procedure

1. After removing the integumentary tissue, the beet root is cut into cubes (cube side 5 mm) and thoroughly washed with water to remove the pigment released from the damaged cells.

2. Drop one piece of beetroot into three test tubes. 5 ml of water are poured into the first and second, 5 ml of a 30% acetic acid solution is poured into the third. The first test tube is left for control. The contents of the second are boiled for 2-3 minutes.

3. The vacuoles of beet root cells contain betacyanin, a pigment that gives color to the root tissue. Tonoplasts of living cells are impenetrable to molecules of this pigment. After cell death, the tonoplast loses its semi-permeable property, becomes permeable, pigment molecules leave the cells and color the water.

In the second and third test tubes, where the cells were killed by boiling or acid, the water becomes colored, but in the first test tube it remains uncolored.

4. Write down the results of your observations.

Laboratory work No. 2

Turgor, plasmolysis and deplasmolysis

Exercise: study under a microscope the phenomena of turgor, plasmolysis and deplasmolysis in the epidermal cells of blue onions.

Materials and equipment: microscopes, dissecting equipment, spirit lamps, blue onions, beet roots, 30% sugar solution, 5-8% potassium nitrate solution.

Operating procedure

1. Make a flat section of the epidermis of a blue onion and place it on a glass slide in a drop of water.

2. Cover the drop with a coverslip and observe the cells in a state of turgor through a microscope.

3. Take a drop of 30% sugar solution and place it next to the cover slip.

4. Touching the filter paper to the opposite end of the cover glass, replace the water in the preparation with a sugar solution.

5. Observe under the microscope again. If plasmolysis is not yet noticeable, repeat replacing the water with a sugar solution.

Under a microscope, plasmolysis in living epidermal cells will be clearly visible.

6. Carry out the experiment in the reverse order, i.e. return the water again and observe the phenomenon of deplasmolysis.

7. Draw cells in a state of turgor, plasmolysis and deplasmolysis.

8. To prove that plasmolysis and deplasmolysis occur only in living cells, carry out such an experiment in parallel. Hold one of the sections of the onion epidermis in a drop of water over the flame of an alcohol lamp to kill the cells. Then apply a sugar solution and see if plasmolysis occurs.

The described experience allows you to get acquainted not only with the processes of turgor, plasmolysis and deplasmolysis, but also with the process of substances entering the cell (in this case, sugar molecules from solution).

When studying the phenomena of plasmolysis and deplasmolysis in beet root cells, the procedure is the same, but instead of a sugar solution it is better to use a 5% solution of potassium nitrate.

Laboratory work No. 3

Determination of transpiration by gravimetric method

Exercise: determine the amount of water evaporated by a plant over a certain period of time using the gravimetric method.

Materials and equipment: scales, weights, scissors, dishes, stand, live plants.

Operating procedure

1. Place the U-shaped tube on a stand and pour water into it. Cut one leaf from the plant (or a small branch with two leaves) and use a cotton plug to secure it in one leg (the cotton plug should not touch the water, otherwise the water will evaporate through it). Close the other elbow with a rubber or plastic stopper (if there is no such tube, you can take a simple test tube and fill the surface of the water with vegetable oil to prevent evaporation).

2. Weigh the device and at the same time a small crystallizer filled with water. Place the device and the crystallizer on the window.

3. After 1-2 hours, re-weigh. The mass decreases in both cases as water evaporates.

Laboratory work No. 4

Observation of stomatal movement

Exercise: observe stomatal movements, explain the reason for stomatal movements, sketch stomata in water and in solutions of 5 and
20%- go glycerin.

Goal of the work: observe stomatal movements in water and in a glycerol solution.

Materials and equipment: glycerin solutions (5 and 20%), 1M sucrose solution, microscopes, slides and cover glasses, dissecting needles, filter paper, bottles, leaves of any plants.

Operating procedure

1. Prepare several sections of the lower epidermis of the leaf and place them in a 5% glycerin solution for 2 hours. Glycerol penetrates the vacuoles of guard cells, lowers their water potential and, therefore, increases their ability to absorb water. The sections are placed on a glass slide in the same solution, the condition of the cells is noted and they are sketched.

2. Replace the glycerin with water, pulling it out from under the glass with filter paper. In this case, the opening of stomatal slits is observed. Draw the drug.

3. Replace the water with a strong osmotic agent - a 20% glycerin solution or a 1M sucrose solution. The closing of stomata is observed.

4. Draw conclusions.

Laboratory work No. 5

Products of photosynthesis

Exercise: study the process of formation of primary starch in leaves.

Materials and equipment: alcohol lamps, water baths, scissors, electric stoves, 200-300 W incandescent lamps, dishes, live plants (pumpkin, beans, pelargonium, primrose, etc.), ethyl alcohol, iodine solution in potassium iodide.

Operating procedure

1. Using a starch test, prove that starch is formed during photosynthesis.

A well-watered plant should be placed in a dark place for 2-3 days. During this time, there will be an outflow of assimilates from the leaves. New starch cannot form in the dark.

To get contrast from the photosynthesis process, part of the leaf must be darkened. To do this, you can use a photo negative or two identical light-proof screens, attaching them to the top and bottom. Pictures on the screen (clippings) can be very different.

An incandescent lamp of 200-300 W is placed at a distance of 0.5 m from the sheet. After an hour or two, the sheet must be processed as indicated above. It is more convenient to do this on a flat plate. At the same time, the sheet that remained darkened all the time is processed.

The parts exposed to light turn blue, while the rest are yellow.

In summer, you can modify the experiment - cover several leaves on the plant, putting bags of black opaque paper with appropriate cutouts on them; after two to three days, at the end of a sunny day, cut off the leaves, boil them first in water, and then bleach them with alcohol and treat them with a solution of iodine in potassium iodide. The darkened areas of the leaves will be light, and the illuminated areas will become black.

In some plants (for example, onions), the primary product of photosynthesis is not starch, but sugar, so the starch test is not applicable to them.

2. Write down the results of your observations.

Laboratory work No. 6

Obtaining pigments from alcoholic extracts of leaves
and their division

Exercise: obtain an alcohol extract of pigments, separate them and become familiar with the basic properties of pigments.

Materials and equipment: scissors, mortars and pestles, racks with test tubes, dishes, alcohol lamps, water baths, fresh or dry leaves (nettle, aspidistra, ivy or other plants), ethyl alcohol, gasoline, 20% NaOH (or KOH) solution, dry chalk , sand.

Operating procedure

1. Place dry leaves crushed with scissors into a clean mortar, add a little chalk to neutralize the acids of the cell sap. Thoroughly grind the mass with a pestle, adding ethyl alcohol (100 cm 3), then filter the solution.

The resulting chlorophyll extract has fluorescence: in transmitted light it is green, in reflected light it is cherry-red.

2. Separate the pigments using the Kraus method.

To do this, you need to pour 2-3 cm3 of extract into a test tube and add one and a half volume of gasoline and 2-3 drops of water; then you need to shake the test tube and wait until two layers become clearly visible - gasoline at the top, alcohol at the bottom. If separation does not occur, add more gasoline and shake the test tube again.

If turbidity appears, add a little alcohol.

Since gasoline does not dissolve in alcohol, it ends up at the top. The green color of the top layer indicates that chlorophyll has transferred into gasoline. In addition to it, carotene also dissolves in gasoline. Below, in the alcohol, xanthophyll remains. The bottom layer is yellow.

After the solution settles, two layers form. As a result of saponification of chlorophyll, alcohols are eliminated and the sodium salt of chlorophyllin is formed, which, unlike chlorophyll, does not dissolve in gasoline.

For better saponification, the test tube with added NaOH can be placed in a water bath with boiling water and, as soon as the solution boils, removed. After this, gasoline is added. Carotene and xanthophyll (the color will be yellow) will go into the gasoline layer (top), and the sodium salt of chlorophyll acid will go into the alcohol layer.

Laboratory work No. 7

Plant respiration detection

Exercise: prove that CO 2 is released when plants respire, sketch a device that helps detect respiration by the release of CO 2, write captions for the drawing.

Materials and equipment: 2 glass jars with a capacity of 300-400 ml, 2 rubber test tubes with holes for a funnel and tube, 2 funnels, 2 glass tubes curved in the shape of the letter “P” 18-20 cm long and 4-5 mm in diameter, 2 test tubes, a beaker, Ba(OH)2 solution, sprouted seeds of wheat, sunflower, corn, peas, etc.

Operating procedure

1. Pour 50-60 g of sprouted seeds into a glass jar, close it tightly with a stopper into which a funnel and a curved glass tube are inserted and leave for 1-1.5 hours. During this time, as a result of the respiration of the seeds, carbon dioxide will accumulate in the jar. It is heavier than air, so it is concentrated at the bottom of the can and does not enter the atmosphere through a funnel or tube.

2. At the same time, take a control jar without seeds, also close it with a rubber stopper with a funnel and a glass tube and place it next to the first jar.

3. The free ends of the glass tubes are lowered into two test tubes with barite water. They begin to gradually pour water into both jars through funnels. Water displaces air enriched with CO 2 from the cans, which enters the test tubes with the Ba(OH) 2 solution. As a result, barite water becomes cloudy.

4. Compare the degree of turbidity of Ba(OH) 2 in both test tubes.

Laboratory work No. 8

Determination of respiration intensity in Conway cups

Exercise: perform an experiment and calculate the breathing intensity of the objects under study depending on the experimental options.

Materials and equipment: Conway cups, Vaseline, burettes, stands, filter paper, scissors, scales, weights, reagents: 0.1 N Ba(OH) 2 ; 0.1 N HCl, phenolphthalein, any seedlings and adult plants or their organs.

Operating procedure

1. Conway cups are calibrated before the experiment; they must be the same volume for the control and experimental variants. Each experimental variant is performed in triplicate.

2. A sample of plant material weighing 0.5-1.0 g is laid out in the outer circle of the Conway cup. 1 or 2 ml of 0.1 N Ba(OH) 2 is poured into the inner cylinder. The cup is hermetically sealed with a ground-in lid (so that on the lid a transparent outline of the thin section of the cup has appeared) and placed in the dark for 20 - 40 minutes (to exclude photosynthesis in green plant tissues). During the exposure, the carbon dioxide accumulated in the Conway cup reacts with barium hydroxide:

CO 2 + Ba(OH) 2 = BaCO 3 + H 2 O.

Excess Ba(OH)2 is titrated with 0.1 N HC1 against phenolphthalein until the pink color disappears.

3. At the same time as the experimental one, place a control Conway cup (without a sample). The same volume of 0.1 N Ba(OH) 2 solution is poured into it, closed with a ground-in lid and left next to the test cup. Barium hydroxide in this cup reacts with carbon dioxide, which was originally contained in the air in its volume. Excess barite is titrated.

4. Based on the difference in the volumes of hydrochloric acid solution used to titrate excess Ba(OH)2 in the control and experimental dishes, the respiration intensity (I.D.) is calculated:

Mg CO 2 /(g∙h),

where V HC1k is the volume of 0.1 N HC1 used for titration of excess Ba(OH) 2 in the control cup; V HC1op - volume of 0.1 N HC1, used for titration of excess Ba(OH) 2 in the test cup; R- sample weight, g;

t - time, h; 2.2 is the conversion factor of HC1 to CO 2 (1 ml of 0.1 N HC1 or Ba(OH) 2 is equivalent to 2.2 mg CO 2).

Laboratory work No. 9

The meaning of various elements for plants

Exercise: study the importance of various mineral elements for the growth of the fungus Aspergillus.

Materials and equipment: scales, thermostat, cotton plugs, filters, five 100 cm 3 flasks, test tubes, pipette, two glasses, funnel, mineral salts, sucrose, organic acid (citric), a culture of the Aspergillus fungus grown on pieces of potato or bread for 3- 4 days.

Operating procedure

1. Grow a mushroom using nutrient mixtures.

It has been established that Aspergillus has approximately the same requirements for mineral nutrition as higher plants. Of the mineral elements, the mushroom does not need only calcium. Nutrient mixtures are prepared in 100 cm 3 flasks and composed according to a specific scheme (Table 1).

The numbering of the flasks corresponds to the numbering of the experimental variants. The results of the experiment are written down below.

Table 1

Scheme for preparing nutritional mixtures

Substances	Concentration	Amount of substance (in ml) in flasks
		No. 1 - complete mixture	No. 2 - without N	No. 3 - without P	No. 4 - without K	No. 5 - without minerals
Sucrose Lemon acid
results Mycelium mass, g

Citric acid is added to create an acidic environment that is favorable for aspergillus, but inhibits the development of other microorganisms.

2. Pour sterile water into a test tube or flask and place the fungal mycelium in it, taken with a sterile loop, stir the contents by rotating between your fingers or palms.

Pipette the resulting suspension into all flasks using a sterile pipette.

Close the flasks with cotton plugs and place in a thermostat at a temperature of 30-35 °C. Observation will be carried out in a week.

The essence of the experiment is that by determining the mass of fungal mycelium grown on various nutrient mixtures, one can find out its need for individual elements.

3. Weigh, for which you take two clean glasses, one funnel and several identical paper filters. Weigh one beaker (No. 1) with a funnel and filter and record the mass. Then place the funnel in another glass (No. 2), transfer the fungal mycelium from the first flask to the filter, rinse with water and after the water flows, transfer the funnel back to glass No. 1. Weigh again. It is clear that the result will be greater, since fungal mycelium has been added.

Educational and methodological manual

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THEORY AND PRACTICE OF SCIENTIFIC SPEECH Special course for non-humanitarian specialties at universities Educational and methodological complex Balashov - 2008

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Educational and methodological complex (219)

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Introduction 2

1.Basic facts about the structure of the cell membrane 3

2. General ideas about permeability 4

3. Transfer of molecules across the membrane 4

3.1. Diffusion 5

3.2 Fick's equation 6

3.3 Passive transport 7

3.3.1 Differences between facilitated diffusion and simple diffusion 8

4. Darcy's Law 8

5. Active transport 9

6. Structure and functions of ion channels 11

Conclusion 15

References 17

INTRODUCTION

Membrane transport is the transport of substances through the cell membrane into or out of the cell, carried out using various mechanisms - simple diffusion, facilitated diffusion and active transport.

The most important property of a biological membrane is its ability to pass various substances into and out of the cell. This is of great importance for self-regulation and maintaining a constant cell composition. This function of the cell membrane is performed due to selective permeability, i.e. the ability to allow some substances to pass through and not others. Nonpolar molecules with low molecular weight (oxygen, nitrogen, benzene) pass most easily through the lipid bilayer. Small polar molecules such as carbon dioxide, nitric oxide, water, and urea penetrate quite quickly through the lipid bilayer. Ethanol and glycerol, as well as steroids and thyroid hormones, pass through the lipid bilayer at a noticeable rate. For larger polar molecules (glucose, amino acids), as well as for ions, the lipid bilayer is practically impermeable, since its interior is hydrophobic. Thus, for water the permeability coefficient (cm/s) is about 10-2, for glycerol – 10-5, for glucose – 10-7, and for monovalent ions – less than 10-10.

The transfer of large polar molecules and ions occurs due to channel proteins or carrier proteins. Thus, in cell membranes there are channels for sodium, potassium and chlorine ions, in the membranes of many cells there are aquaporin water channels, as well as carrier proteins for glucose, various groups of amino acids and many ions. Active and passive transport.

Membranes form the structure of the cell and carry out its functions. Disruption of the functions of cellular and intracellular membranes underlies irreversible cell damage and, as a consequence, the development of severe diseases of the cardiovascular, nervous, and endocrine systems.

1. Basic facts about the structure of the cell membrane.

Cell membranes include plasma membrane, karyolemma, membranes of mitochondria, ER, Golgi apparatus, lysosomes, and peroxisomes. A common feature of all cell membranes is that they are thin (6-10 nm) layers of lipoprotein nature (lipids in complex with proteins). The main chemical components of cell membranes are lipids (40%) and proteins (60%); in addition, carbohydrates (5-10%) were found in many membranes.

The plasma membrane surrounds each cell, determines its size, and maintains the distinction between the cell's contents and its external environment. The membrane serves as a highly selective filter and is responsible for the active transport of substances, that is, the entry of nutrients into the cell and the removal of harmful waste products. Finally, the membrane is responsible for the perception of external signals and allows the cell to respond to external changes. All biological membranes are assemblies of lipid and protein molecules held together by non-covalent interactions.

The basis of any molecular membrane is made up of lipid molecules that form a bilayer. Lipids include a large group of organic substances that have poor solubility in water (hydrophobicity) and good solubility in organic solvents and fats (lipophilicity). The composition of lipids in different membranes is not the same. For example, the plasma membrane, unlike the membranes of the endoplasmic reticulum and mitochondria, is enriched in cholesterol. Typical representatives of lipids found in cell membranes are phospholipids (glycerophosphatides), sphingomyelins and steroid lipids - cholesterol.

A feature of lipids is the division of their molecules into two functionally different parts: hydrophobic non-polar, non-charge-bearing (“tails”), consisting of fatty acids, and hydrophilic, charged polar “heads”. This determines the ability of lipids to spontaneously form bilayer (bilipid) membrane structures with a thickness of 5-7 nm.

The first experiments confirming this were carried out in 1925.

The formation of a bilayer is a special property of lipid molecules and occurs even outside the cell. The most important properties of a bilayer: ability to self-assemble - fluidity - asymmetry.

2. General ideas about permeability.

Characteristics of membranes, vessel walls and epithelial cells, reflecting the ability to conduct chemicals; distinguish between active (active transport of substances) and passive P. (phagocytosis And pinocytosis ); passive and (in some cases) active P. (large molecules) are provided by membrane pores; P. for low-molecular substances (for example, ions) is provided by specific membrane structures with the participation of carrier molecules.

3. Transfer of molecules across the membrane.

Since the interior of the lipid layer is hydrophobic, it represents a virtually impenetrable barrier to most polar molecules. Due to the presence of this barrier, leakage of cell contents is prevented, but because of this, the cell was forced to create special mechanisms to transport water-soluble substances across the membrane. The transfer of small water-soluble molecules is carried out using special transport proteins. These are special transmembrane proteins, each of which is responsible for the transport of specific molecules or groups of related molecules.

Cells also have mechanisms for transporting macromolecules (proteins) and even large particles across the membrane. The process of uptake of macromolecules by a cell is called endocytosis. In general terms, the mechanism of its occurrence is as follows: local areas of the plasma membrane are invaginated and closed, forming an endocytic vesicle, then the absorbed particle usually enters lysosomes and undergoes degradation.

3.1 Diffusion (Latin diffusio - spreading, spreading, dispersing) is the process of transferring matter or energy from an area of ​​high concentration to an area of ​​low concentration (against the concentration gradient). The most famous example of diffusion is the mixing of gases or liquids (if ink is dropped into water, the liquid will become uniformly colored after some time). Another example involves a solid: if one end of a rod is heated or electrically charged, heat (or, correspondingly, electric current) spreads from the hot (charged) part to the cold (uncharged) part. In the case of a metal rod, thermal diffusion develops quickly and the current flows almost instantly. If the rod is made of a synthetic material, thermal diffusion is slow and diffusion of electrically charged particles is very slow. Diffusion of molecules is generally even slower. For example, if a piece of sugar is placed at the bottom of a glass of water and the water is not stirred, it will take several weeks before the solution becomes homogeneous. Diffusion of one solid substance into another occurs even more slowly. For example, if copper is coated with gold, then diffusion of gold into the copper will occur, but under normal conditions (room temperature and atmospheric pressure) the gold-bearing layer will reach a thickness of several micrometers only after several thousand years.

All types of diffusion obey the same laws. The rate of diffusion is proportional to the cross-sectional area of ​​the sample, as well as the difference in concentrations, temperatures or charges (in the case of relatively small values ​​of these parameters). Thus, heat will spread four times faster through a rod with a diameter of two centimeters than through a rod with a diameter of one centimeter. This heat will spread faster if the temperature difference across one centimeter is 10°C instead of 5°C. The rate of diffusion is also proportional to the parameter characterizing a particular material. In the case of thermal diffusion, this parameter is called thermal conductivity; in the case of the flow of electric charges, it is called electrical conductivity. The amount of substance that diffuses over a given time and the distance traveled by the diffusing substance are proportional to the square root of the diffusion time.

Diffusion is a process at the molecular level and is determined by the random nature of the movement of individual molecules. The rate of diffusion is therefore proportional to the average speed of the molecules. In the case of gases, the average speed of small molecules is greater, namely, it is inversely proportional to the square root of the mass of the molecule and increases with increasing temperature. Diffusion processes in solids at high temperatures often find practical application. For example, certain types of cathode ray tubes (CRTs) use thorium metal diffused through tungsten metal at 2000 °C.

3.2 Fick's equation

In most practical cases, the concentration C is used instead of the chemical potential. Direct replacement of µ by C becomes incorrect in the case of high concentrations, since the chemical potential is related to concentration according to a logarithmic law. If we do not consider such cases, then the above formula can be replaced with the following:

which shows that the substance flux density J is proportional to the diffusion coefficient D and the concentration gradient. This equation expresses Fick's first law (Adolph Fick is a German physiologist who established the laws of diffusion in 1855). Fick's second law relates spatial and temporal changes in concentration (diffusion equation):

The diffusion coefficient D depends on temperature. In a number of cases, over a wide temperature range, this dependence represents the Arrhenius equation.

Diffusion processes are of great importance in nature:

Nutrition, respiration of animals and plants;

Penetration of oxygen from blood into human tissues.

3.3 Passive transport

Passive transport is the transfer of substances from places with a high electrochemical potential to places with a lower value.

In experiments with artificial lipid bilayers, it was found that the smaller the molecule and the fewer hydrogen bonds it forms, the faster it diffuses through the membrane. So, the smaller the molecule and the more fat-soluble (hydrophobic or non-polar) it is, the faster it will penetrate the membrane. Diffusion of substances across the lipid bilayer is caused by a concentration gradient in the membrane. Molecules of lipid-insoluble substances and water-soluble hydrated ions (surrounded by water molecules) penetrate the membrane through lipid and protein pores. Small nonpolar molecules are easily soluble and diffuse quickly. Uncharged polar molecules with small sizes are also soluble and diffuse.

It is important that water penetrates the lipid bilayer very quickly despite the fact that it is relatively insoluble in fats. This is due to the fact that its molecule is small and electrically neutral.

Osmosis is the preferential movement of water molecules through semipermeable membranes (impermeable to solute and permeable to water) from places of lower solute concentration to places of higher concentration. Osmosis is essentially the simple diffusion of water from places with a higher concentration of water to places with a lower concentration of water. Osmosis plays a large role in many biological phenomena. The phenomenon of osmosis causes hemolysis of red blood cells in hypotonic solutions.

So, membranes can allow water and non-polar molecules to pass through through simple diffusion.

3.3.1 Differences between facilitated diffusion and simple:

1) transfer of a substance with the participation of a carrier occurs much faster;

2) facilitated diffusion has the property of saturation: with increasing concentration on one side of the membrane, the flux density of the substance increases only to a certain limit, when all the carrier molecules are already occupied;

3) with facilitated diffusion, competition between transported substances is observed in cases where the carrier transports different substances; Moreover, some substances are better tolerated than others, and the addition of some substances complicates the transport of others; Thus, among sugars, glucose is better tolerated than fructose, fructose is better than xylose, and xylose is better than arabinose, etc. etc.;

4) there are substances that block facilitated diffusion - they form a strong complex with carrier molecules, for example, phloridzin inhibits the transport of sugars through a biological membrane.

4.Darcy's Law

Darcy's Law (Henri Darcy, 1856) - the law of filtration of liquids and gases in a porous medium. Obtained experimentally. Expresses the dependence of the fluid filtration rate on the pressure gradient:

where: - filtration rate, K - filtration coefficient, - pressure gradient. Darcy's law is associated with several measurement systems. A medium with a permeability of 1 Darcy (D) allows the flow of 1 cm³/s of liquid or gas with a viscosity of 1 cp (mPa s) under a pressure gradient of 1 atm/cm acting over an area of ​​1 cm². 1 millidarcy (mD) is equal to 0.001 Darcy.

In the SI measurement system, 1 Darcy is equivalent to 9.869233×10−13 m² or 0.9869233 µm². This conversion is usually approximated as 1 µm². It should be noted that this number is the reciprocal of 1.013250 - the conversion factor from atmospheres to bars.

Transport through the lipid bilayer (simple diffusion) and transport with the participation of membrane proteins

5. Active transport

Other carrier proteins (sometimes called pump proteins) transport substances across the membrane using energy, which is usually supplied by the hydrolysis of ATP. This type of transport occurs against the concentration gradient of the transported substance and is called active transport.

Simport, antiport and uniport

Membrane transport of substances also differs in the direction of their movement and the amount of substances carried by a given carrier:

1) Uniport - transport of one substance in one direction depending on the gradient

2) Symport - transport of two substances in one direction through one carrier.

3) Antiport - movement of two substances in different directions through one carrier.

Uniport carries out, for example, a voltage-gated sodium channel through which sodium ions move into the cell during the generation of an action potential.

Symport is carried out by a glucose transporter located on the external (facing the intestinal lumen) side of the intestinal epithelial cells. This protein simultaneously captures a glucose molecule and a sodium ion and, changing conformation, transfers both substances into the cell. This uses the energy of the electrochemical gradient, which in turn is created due to the hydrolysis of ATP by sodium-potassium ATPase.

Antiport is carried out, for example, by sodium-potassium ATPase (or sodium-dependent ATPase). It transports potassium ions into the cell. and from the cell - sodium ions.

The work of sodium-potassium ATPase as an example of antiport and active transport

Initially, this transporter attaches three Na + ions to the inner side of the membrane. These ions change the conformation of the active site of ATPase. After such activation, the ATPase is able to hydrolyze one ATP molecule, and the phosphate ion is fixed on the surface of the transporter on the inside of the membrane.

The released energy is spent on changing the conformation of the ATPase, after which three Na + ions and an ion (phosphate) end up on the outside of the membrane. Here, Na + ions are split off and replaced by two K + ions. Then the conformation of the carrier changes to its original one, and K + ions end up on the inner side of the membrane. Here the K+ ions are split off and the transporter is ready for use again.

More briefly, the actions of ATPase can be described as follows:

1) It “takes” three Na + ions from inside the cell, then splits the ATP molecule and adds phosphate to itself

2) “Throws out” Na + ions and attaches two K + ions from the external environment.

3) Disconnects phosphate, releasing two K+ ions into the cell

As a result, a high concentration of Na + ions is created in the extracellular environment, and a high concentration of K + ions is created inside the cell. The work of Na +, K + - ATPase creates not only a concentration difference, but also a charge difference (it works like an electrogenic pump). A positive charge is created on the outside of the membrane, and a negative charge on the inside.

6. Structure and functions of ion channels.

The excitable membrane model assumes the regulated transport of potassium and sodium ions across the membrane. However, the direct passage of an ion through the lipid bilayer is very difficult, so the ion flux density would be very low if the ion passed directly through the lipid phase of the membrane. This and a number of other considerations gave reason to believe that the membrane must contain some special structures - conducting ions.

Such structures were found and called ion channels. Similar channels have been isolated from various objects: the plasma membrane of cells, the postsynaptic membrane of muscle cells and other objects. Ion channels formed by antibiotics are also known.

Basic properties of ion channels:

1) selectivity;

2) independence of operation of individual channels;

3) discrete nature of conductivity;

4) dependence of channel parameters on membrane potential.

Let's look at them in order.

1. Selectivity is the ability of ion channels to selectively allow ions of one type to pass through.

Even in the first experiments on the squid axon, it was discovered that sodium and potassium ions have different effects on the membrane potential. Potassium ions change the resting potential, and sodium ions change the action potential.

Measurements have shown that ion channels have absolute selectivity towards cations (cation-selective channels) or anions (anion-selective channels). At the same time, various cations of various chemical elements can pass through cation-selective channels, but the conductivity of the membrane for the minor ion, and therefore the current through it, will be significantly lower, for example, for the sodium channel, the potassium current through it will be 20 times less. The ability of an ion channel to pass different ions is called relative selectivity and is characterized by a selectivity series - the ratio of channel conductivities for different ions taken at the same concentration.

2. Independence of the operation of individual channels. The flow of current through an individual ion channel is independent of whether current flows through other channels. For example, potassium channels can be turned on or off, but the current through sodium channels does not change. The influence of channels on each other occurs indirectly: a change in the permeability of some channels (for example, sodium) changes the membrane potential, and this already affects the conductivity of other ion channels.

3. Discrete nature of the conductivity of ion channels. Ion channels are a subunit complex of proteins that span the membrane. In its center there is a tube through which ions can pass.

The number of ion channels per 1 μm of membrane surface was determined using a radiolabeled sodium channel blocker, tetrodotoxin. It is known that one TTX molecule binds to only one channel. Then measuring the radioactivity of a sample with a known area made it possible to show that there are about 500 sodium channels per 1 micron of squid axon. This was first discovered in 1962 in studies of the conductivity of lipid bilayer membranes (BLMs) when microquantities of a certain excitation-inducing substance were added to the solution surrounding the membrane. A constant voltage was applied to the BLM and the current was recorded. The current was recorded over time in the form of jumps between two conducting states.

The results of experiments performed on various ion channels showed that the conductivity of an ion channel is discrete and it can be in two states: open or closed. Current surges are caused by the simultaneous opening of 2 or 3 channels. Transitions between states of the ion channel occur at random times and obey statistical laws. It cannot be said that a given ion channel will open at exactly this moment in time. You can only make a statement about the probability of opening a channel in a certain time interval.

Ion channels are described by the characteristic lifetimes of the open and closed states.

4. Dependence of channel parameters on membrane potential. Nerve fiber ion channels are sensitive to membrane potential, such as the sodium and potassium channels of the squid axon. This is manifested in the fact that after the start of membrane depolarization, the corresponding currents begin to change with one or another kinetics. In the language of “ion channels,” this process occurs as follows. The ion-selective channel has a so-called

“sensor” is a certain element of its design that is sensitive to the action of an electric field (see figure). When the membrane potential changes, the magnitude of the force acting on it changes, as a result, this part of the ion channel moves and changes the probability of opening or closing the “gate” - a kind of damper that operates according to the “all or nothing” law.

Ion channel structure

The ion-selective channel consists of the following parts, a protein part immersed in the bilayer, which has a subunit structure; a selective filter formed by negatively charged oxygen atoms, which are rigidly located at a certain distance from each other and allow ions of a certain diameter to pass through; gate part.

The “gate” of the ion channel is controlled by the membrane potential and can be in either a closed state (dashed line) or an open state (solid line). The normal position of the sodium channel gate is closed. Under the influence of an electric field, the probability of an open state increases, the gate opens and the flow of hydrated ions is able to pass through the selective filter.

If the ion “fits” in diameter, then it sheds the hydration shell and jumps to the other side of the ion channel. If the ion is too large in diameter, such as tetraethylammonium, it is not able to fit through the filter and cannot cross the membrane. If, on the contrary, the ion is too small, then it has difficulties in the selective filter, this time associated with the difficulty of shedding its hydration shell. For the “suitable” ion, the discarded water is replaced by bonds with oxygen atoms located in the filter; for the “unsuitable” ion, the steric fit is worse. Therefore, it is more difficult for it to pass through the filter and the channel conductivity is lower for it.

Ion channel blockers either cannot pass through it, getting stuck in the filter, or, if they are large molecules like TTX, they sterically match some entrance to the channel. Since blockers carry a positive charge, their charged part is drawn into the channel to the selective filter as an ordinary cation, and the macromolecule clogs it.

Thus, changes in the electrical properties of excitable biomembranes are carried out using ion channels. These are protein macromolecules that penetrate the lipid bilayer and can exist in several discrete states. The properties of channels selective for potassium, sodium and calcium ions may depend differently on the membrane potential, which determines the dynamics of the action potential in the membrane, as well as the differences in such potentials in the membranes of different cells.

Conclusion

Any molecule can pass through the lipid bilayer, but the rate of passive diffusion of substances, i.e. The transition of a substance from an area of ​​higher concentration to an area of ​​lower concentration can be very different. For some molecules this takes such a long time that we can talk about their practical impermeability to the lipid bilayer of the membrane. The rate of diffusion of substances through a membrane depends mainly on the size of the molecules and their relative solubility in fats.

Small nonpolar molecules such as O2, steroids, thyroid hormones, and fatty acids pass most easily through simple diffusion through the lipid membrane. Small polar uncharged molecules - CO2, NH3, H2O, ethanol, urea - also diffuse at a fairly high speed. The diffusion of glycerol is much slower, and glucose is practically unable to pass through the membrane on its own. The lipid membrane is impermeable to all charged molecules, regardless of size.

The transport of such molecules is possible due to the presence in the membranes of either proteins that form channels (pores) in the lipid layer filled with water, through which substances of a certain size can pass by simple diffusion, or specific carrier proteins that, selectively interacting with certain ligands, facilitate their transport through membrane (facilitated diffusion).

In addition to passive transport of substances, cells contain proteins that actively pump certain substances dissolved in water against their gradient, i.e. from a lower concentration to an area of ​​higher concentration. This process, called active transport, is always carried out with the help of carrier proteins and occurs with the expenditure of energy.

The outer part of the canal is relatively accessible for study; studying the inner part presents significant difficulties. P. G. Kostyuk developed a method of intracellular dialysis, which allows one to study the function of the input and output structures of ion channels without the use of microelectrodes. It turned out that the part of the ion channel open to the extracellular space differs in its functional properties from the part of the channel facing the intracellular environment.

It is ion channels that provide two important properties of the membrane: selectivity and conductivity.

The selectivity, or selectivity, of the channel is ensured by its special protein structure. Most channels are electrically controlled, that is, their ability to conduct ions depends on the magnitude of the membrane potential. The channel is heterogeneous in its functional characteristics, especially with regard to the protein structures located at the entrance to the channel and at its exit (the so-called gate mechanisms).

Fick's equation

The “–” sign shows that the total flux density of a substance during diffusion is directed towards decreasing density, D is the diffusion coefficient. The formula shows that the flux density of a substance J is proportional to the diffusion coefficient D and the concentration gradient. This equation expresses Fick's first law (Adolph Fick is a German physiologist who established the laws of diffusion in 1855).

The ion-selective channel consists of the following parts, a protein part immersed in the bilayer, which has a subunit structure; a selective filter formed by negatively charged oxygen atoms, which are rigidly located at a certain distance from each other and allow ions of a certain diameter to pass through; gate part. It is ion channels that provide two important properties of the membrane: selectivity and conductivity. Calcium channels play an essential role in heart cells.

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