Blood and erythrocytes. We continue to publish materials about blood.
What does an erythrocyte look like? Under normal physiological conditions in the bloodstream, erythrocytes have a biconcave shape with uniform thickening along the edges and with a central lighter part - pellor.
In a light-optical study, a normal erythrocyte routinely stained with acid dyes has the shape of a disk with a diameter of 6.9-7.7 and up to 9.0 microns. Depending on the size, erythrocytes are divided into micro- and macrocytes, but most of them are represented by normocytes / discocytes.
Morphofunctional properties of an erythrocyte
An erythrocyte is a nuclear-free biconcave cell with an average volume of 90.0 µm 3 and an area of 142 µm 2 . Its greatest thickness is 2.4 µm, the minimum is 1 µm.
In the dried preparation, the average size of an erythrocyte is 7.55 µm; 95% of its dry matter falls on the iron-containing protein hemoglobin and only 5% - on the share of other substances (other proteins and lipids). Such cells represent the absolute majority - over 85% - of healthy human erythrocytes.
The nuclear forms of an erythrocyte germ are easily distinguished from most cells of the leukocyte series by the absence of granules in their cytoplasm (errors are possible only when identifying blast cells). Erythroblasts are characterized by more granular and denser nuclear chromatin.
The central cavity (pellor) of the erythrocyte disk accounts for 35 to 55% of its surface, and on the cross section, the erythrocyte has the shape of a donut, which, on the one hand, ensures the preservation of hemoglobin and, on the other hand, allows the erythrocyte to pass through even the thinnest capillaries. The currently available models of the erythrocyte structure correspond to the concept of the specific properties of this cell, especially its membrane, which, despite its sensitivity to deforming pressure, provides resistance to bending and an increase in the total surface.
Literature data indicate that the size and deformability of the erythrocyte membrane are their most important characteristics, which are associated with the normal functioning of these cells, including high migration ability, participation in metabolic processes (primarily in oxygen exchange).
Changes in the microelastometric properties of erythrocytes and the "transformation" of discocytes into other morphological forms can be caused by various agents. Thus, the appearance of superficial outgrowths leads to a decrease in the elasticity of the membrane, which may be due to opposite forces that arise in the very process of erythrocyte deformation; deformation increases with a decrease in the concentration of ATP in the cells.
If the integrity of the cell membrane is violated, then the erythrocyte loses its characteristic shape and turns into a spheroplast, which, in turn, is hemolyzed. The structure of the erythrocyte membrane (discocyte) is the same throughout; and despite the fact that depressions and bulges can occur in its various parts, changes in intra- or extracellular pressure with a spread of ± 15% do not cause wrinkling of the entire cell, because it has a significant margin of "anti-deformability". The erythrocyte membrane has sufficient elasticity to withstand the influence of various factors that occur during the circulation of the erythrocyte through the bloodstream.
The composition of the erythrocyte membrane includes: phospholipids (36.3%), sphingomyelins (29.6%), cholesterol (22.2%) and glycolipids (11.9%). The first two elements are amphiphilic molecules in an aqueous medium, forming a characteristic lipid bilayer, which is also permeated with integral protein molecules associated inside the erythrocyte with its cytoskeleton.
Membrane lipids are in a liquid state, have a low viscosity (only 10-100 times the viscosity of water). Lipids, sialic acid, antigenic oligosaccharides, adsorbed proteins are located on the outer surface of the membrane; the inner surface of the membrane is represented by glycolytic enzymes, sodium and calcium, ATPase, glycoproteins and hemoglobin.
The double lipid layer of the membrane performs three functions: the function of a barrier for ions and molecules, the structural basis for the functioning of receptors and enzymes (proteins, glycoproteins, glycolipids) and mechanical. In the implementation of a specialized, respiratory function - the transfer of oxygen or carbon dioxide - the main role is played by membrane proteins "embedded" in the lipid bilayer. Mature erythrocytes are not capable of synthesizing nucleic acids and hemoglobin; they are characterized by a low level of metabolism, which ensures a sufficiently long period of life of these cells (120 days).
As the erythrocyte ages, its surface area decreases, while the hemoglobin content remains unchanged. It has been established that in the "mature" age, erythrocytes maintain a constant chemical composition for a long time, but as the cells age, the content of chemicals in them gradually decreases. The erythrocyte cytoskeleton is formed and controlled by multigene and membrane-associated "families" of proteins that organize specialized membrane domains that maintain the function and shape of this highly specialized cell.
Electric potential of the erythrocyte
The erythrocyte membrane contains 50% protein, up to 45% lipids and up to 10% carbohydrates. On the surface of intact cells, the "network" distribution of charges is determined by a glycoprotein containing sialic (neutramic) acid, which determines up to 62% of the surface negative charge of the cell.
It is believed that each electrical charge corresponds to 1 molecule of this acid. The loss of sialic acid by the erythrocyte surface leads to a decrease in its electrophoretic mobility (EPM) and suppression of cation transport. Consequently, there is a "mosaic" of charges on the cell surface, determined by cationic and anionic groups, the ratio of which determines the total electrical charge of erythrocytes.
To maintain an optimal state of homeostasis, blood cells must have a stable charge. The high stability of EFP is ensured by a subtle mechanism of its regulation - the balance of lipid peroxidation (LPO) processes in erythrocyte membranes and the protective effect of the antioxidant system.
It has been empirically established that receptors for antibodies are located on the erythrocyte membrane, and the presence of even a small amount of them on the surface can disrupt normal physiological functions in the body and change the EFP of erythrocytes. This may affect the level of hemoglobin in the latter, since the content of hemoglobin and EFP is strictly coordinated.
It should also be taken into account that under extreme effects of negative factors on the body, products of lipid peroxidation affect the electrokinetic properties of erythrocytes. In turn, this is reflected in the rate of peroxide processes in their membranes.
Thanks to the electrostatic repulsion (“spread” according to Chizhevsky) of like-charged erythrocyte cells, the latter move freely through the blood vessels, performing their oxygen-transport function. Therefore, a violation of charge stability can be considered an integral indicator of pathological changes in the body.
1.5. Topics of practical classes
SECTION 1. MEMBRANE BIOPHYSICS
1. 1. Biological membranes. Structure, properties.
The specific electrical capacitance of the axon membrane, measured with an intracellular microelectrode, was found to be 0.5 microfarad/cm2. Using the flat capacitor formula, estimate the thickness of the hydrophobic layer of a membrane with a dielectric constant of 2.
What is the distance on the surface of the erythrocyte membrane that a phospholipid molecule travels in 1 second as a result of lateral diffusion? The coefficient of lateral diffusion is taken equal to 10 -12 m 2 /s. Compare with the circumference of an erythrocyte with a diameter of 8 microns.
During the phase transition of membrane phospholipids from the liquid-crystalline state to the gel, the thickness of the bilayer changes. How will the electrical capacitance of the membrane change in this case? How will the electric field strength in the membrane change?
With the help of spin-labeled phospholipid molecules, the viscosity gradient across the membrane thickness was established. Describe the ex-experiment. Where is the viscosity higher: at the surface of the membrane or in its center?
1.1.1. Biological membrane thickness:
10 A, 3. OD µm
10 nm 4. 10 µm
1.1.2. The fluid mosaic model of a biological membrane includes:
protein layer, polysaccharides and surface lipids
lipid monolayer and cholesterol
lipid bilayer, proteins, microfilaments
lipid bilayer
1.1.3. The lipid part of the biological membrane is in the following physical state:
liquid amorphous
solid crystalline
solid amorphous
liquid crystal
1.1.4. Specific electrical capacitance of the axon membrane:
1.1.5. The characteristic transfer time of the transfer of a phospholipid molecule from one equilibrium position to another during their diffusion:
1.1.6. The phase transition of the lipid bilayer of membranes from the liquid-crystalline state to the gel is accompanied by:
membrane thinning
membrane thickness does not change
membrane thickening
1.2. Transport of substances across biological membranes.
Control questions, tasks, assignments for seminars
1. What parameters does the critical radius of a lipid pore in a membrane depend on?
2. Calculate the critical pore radius in the absence of a membrane potential. Take the edge tension of the pore 10 -11 N, the surface tension of the lipid bilayer 0.3 mN/m.
3. How will the facilitated diffusion of kaoium ions with the participation of the valinomycin molecule change after the phase transition of membrane lipids from liquid-crystalline states to gel?
4. The specific electrical capacitance of the axon membrane, measured with an intracellular microelectrode, turned out to be 0.5 microfarad/cm2. Using the flat capacitor formula, estimate the thickness of the hydrophobic layer of a membrane with a dielectric constant of 2.
Model monitoring tests
1.2.1. Ion transport occurs in the direction:
1.2.2. The diffusion equation for non-electrolytes (Fika) is written:
2.3. The valinomycin molecule transports across the membrane:
1.2.4. The transfer of matter during facilitated diffusion is compared with simple diffusion:
slower
1.3. Bioelectric potentials.
Control questions, tasks, assignments for seminars
What transport of ions creates a membrane potential difference: passive or active?
Which is greater: the speed of propagation of an electrical signal along the wires of a marine telegraph or the speed of propagation of a nerve impulse along an axon membrane? Why?
Explain the biophysical mechanism of action of the poison Tetro-Dotoxin and the local anesthetic tetraethylammonium.
How do the permeability of the squid axon membrane for various ions at rest and during excitation correlate?
How will the form of the action potential graph change if we change the chemical composition inside the axon and outside: axo-plasma is replaced with extracellular fluid, and extracellular fluid - with axoplasm?
What is the electric field strength on the membrane at rest, if the concentration of potassium ions inside the cell is 125 mmol / l, outside - 2.5 mmol / l, and the membrane thickness is 8 nm?
(Answer: 1.3 * 10 7 V / m.)
7. Calculate the amplitude of the action potential, if con-
concentration of potassium and sodium inside the cell of excitable tissue
neither respectively: 125 mmol / l, 1.5 mmol / l, and outside
2.5 mmol/l and 125 mmol/l.(Answer: 160 mV.)
Model monitoring tests
1.3.1. Membrane potential f m is called:
1.3.2. Diameter of the tip of the intracellular electrode used for membrane potential measurements:
commensurate with cell size
much smaller than the cell
much larger cells
1.4. Action potential generation mechanism.
Control questions, tasks, assignments for seminars
1. Is it possible for a process on the membrane of an excitable cell, in which flows of different ions with the same charge sign simultaneously flow towards?
2. What is the meaning of the expression
for phase II of the cardiomyocyte action potential?
3. What is the reason that the current through the channel is discrete, and through the membrane - continuous, smoothly changing?
Model monitoring tests
1.4.1. In the depolarization phase during excitation of the axon, the flows of Na + ions are directed:
1. hell 2. bd 3. hell 4. in 5. ag
1. 4.2. In the axon repolarization phase, ion flows are directed:
1.ad 2.bd 3.be 4.d
4.3. Cardiomyocyte action potential duration compared to axon action potential
1. greater than 2. less than 3. equal
4.4. The plateau phase in a cardiomyocyte is determined by ion fluxes:
1. Antonov V.F. Biophysics of membranes // Sorovsky educational journal. - 1997. - T. - 6. S. 1-15.
2. Antonov V.F., Smirnova E.Yu., Shevchenko E.V. Lipid membranes during phase transformations. - M.: Nauka, 1992. - S. 125.
3. Klenchin V.A. biological membranes. - 1993. - T. 10. -S. 5-19.
4. Chizmajaev Yu.A., Arakelyan V.B., Pastushenko V.F. Biophysics of membranes. - M.: Nauka, 1981. - S. 207-229.
5. Kotek A., Janacek K. membrane transport. M.: Mir, 1980.
6. Lightfoot E. Transport phenomena in living systems. M.: 1977.
7. Rubin A.B. Biophysics. M.: Higher. Shk., 1987.
8. Biological membranes: Collection / Under. Ed. D.S. Parsons. Moscow: Atomizdat, 1978.
9. Membrane: Ion channels: Sat. Art. M.: Mir, 1981.
10. Hills B.V. Sat. Membrane: ion channels. M.: Mir, 1981.
11. Physiology and pathophysiology of the heart. Under. ed. N. Sperelakis: M.: Medicine, 1998.
12. Human physiology. Under. ed. Schmidt R. And Tevs G. T. 1. M .: Mir, 1996.
SECTION 2. BIOPHYSICS OF CELLS AND ORGANS
2. 1. Electrical activity of organs.
Control questions, tasks, assignments for seminars
1. What is the principle of an equivalent generator? Give examples of the use of this principle.
2. Why is the inverse problem of electrocardiography a diagnostic task, and not a direct one?
3. What is the mechanism for the formation of a map of electrical potentials on the surface of the human body?
4. Why is it necessary to record at least 3 ECG leads, and not, for example, one?
Model monitoring tests
2.1.1. When modeling ECG, it is assumed that the environment surrounding the dipoles
a. homogeneous a, heterogeneous
b. isotropic b", anisotropic
in. limited in", infinite
1. abc 2. a"b"c" 3.ab"c 4.abc"
2.1.2. What is the reason for changes in the magnitude and direction of the integral electric vector of the heart during the cycle of its work?
contraction of the ventricles of the heart
successive coverage of the wave of excitation of various structures of the heart
metabolic activity of cardiomyocytes
slowing down the speed of the wave in the atrioventricular node
2.1.3. Why are the amplitudes of the same ECG teeth at the same time in different leads not the same?
for different leads, the value of the integral electric vector E _
in different leads, the rotation of the vector E is different
the projections of the vector E on different leads are not the same
each lead has its own vector E
2.1.4. The integral electrical vector of the heart E describes the loops P, QRS, T:
1.horizontal
2.in the plane of the surface of the chest
Z.in volume space XYZ
4. in the plane connecting the points of the right, left hand and left leg
2.1.5 Recorded potential differences
1. ag 2. be 3. vg 4. dv
2.2. Autowave processes in active media.
Control questions, tasks, assignments for seminars
What is the fundamental difference between autowaves in active media and mechanical waves in elastic media?
Why does an autowave propagate in an active medium without damping?
Is interference of auto-waves observed in active media?
What do the autowave parameters in the active medium depend on?
The threshold potential for the cells of the myocardial region is - 30 mV. The transmembrane potential of the cells in this area at some point in time reached a value of 40 mV. Can an excitation wave be transmitted through this area of the myocardium?
Model monitoring tests
2.2.1. The excitation wave (autowave), propagating through the active medium (for example, through the structure of the myocardium), does not decay:
by transferring energy from one cell to another
will detect the release of energy stored by each cell
as a result of the transfer of mechanical energy of myocardial contraction
as a result of using the energy of the electric field
2.2.2 The excitation wavelength in the active medium depends on:
a. amplitudes of the action potential of the cardiomyocyte
b. on the speed of wave propagation through the myocardium
in. on the pulse frequency of the pacemaker
g. from the duration of the refractory period of the excited
cells1. ab 2. bg 3. cg 4. ag
2.2.3. The circulation of an autowave (reentry) of duration X in a ring with a perimeter / can occur under the condition:
2.2.4. If in an inhomogeneous active medium there are zones with refractoriness R 1 and R 2 (R 2 > R:) and impulses from the pacemaker follow with a period T, then rhythm transformation can occur under the condition:
1. T
R 1 3.T = R 2 -R 1 2.3. Biophysics of muscle contraction.
Control questions, tasks, assignments for seminars
Why does the isometric contraction have a different form of dependence F(t) at different initial muscle lengths?
Is it possible to determine the maximum load a muscle can hold from the V(P) Hill curve?
Does the efficiency of muscle contraction increase with increased heat generation by that muscle?
What are the differences between electromechanical coupling in cardiomyocyte and skeletal muscle?
Model monitoring tests
2.3.1. During muscle contraction:
a. actin filaments slide into the sarcomere along the myosin
b. myosin compresses like a spring
in. bridges attach to actin active sites
d. bridges open
1. av 2. bg 3. bv 4. ag
2.3.2. The force of contraction generated by a muscle is determined by:
1. active thread length
2 change in force generated by one bridge
the number of simultaneously closed bridges
elasticity of the myosin filament
2.3.3. The dependence of the speed v of a single muscle contraction on the load P has the form:
2.3.4. Electromechanical coupling is determined by the following chain of events:
a. release of Ca 2+ ions on myofibrils
b. excitation of the cell membrane
in. active transport of Ca 2+ ions into the sarcoplasmic reticulum
d. closure of bridges to actin active centers
e. glide of actin into the sarcomere
1. Human physiology. T. 2. M.: Mir, 1996.
2. Vasiliev V.A., Romanovsky Yu.N., Yakhno V.G. Autowave processes. Moscow: Nauka, 1987.
3.Ivanitsky G.R., Krinsky V.I., Selkov E.E. Mathematical biophysics of the cell. Moscow: Nauka, 1978.
4. Chernysh A.M. Biomechanics of inhomogeneities of the heart muscle. Moscow: Nauka, 1993.
5. Bendol J. Muscles, molecules and movement. M.: Mir, 1989.
SECTION 3. BIOPHYSICS OF COMPLEX SYSTEMS
3.1. Modeling of biophysical processes.
Control questions, tasks, assignments for seminars
How long after the injection will 10% of the initial mass of the drug remain in the blood if the excretion constant k = 0.3 (1/hour)?
The excretion constants of two different drugs differ by a factor of two. Draw qualitative graphs of changes in the mass of the drug in the blood during injections for these two cases. How many times do the excretion rates differ at t = O?
Some time after the patient was put on a drip (when the concentration of the drug reached the stationary level), he was given an injection. Draw a qualitative graph of the change in the mass of the drug over time.
Model monitoring tests
3.1.1. The predator-prey model shows that the populations of predators and prey perform harmonic oscillations. Are the frequencies and phases of these oscillations the same?
a. frequencies are the same. the phases are the same
b. frequencies are different d. phases are different
1. av 2. bv 3. ag 4. bg
3.1.2. What model is adequate for studies of electrogenesis in cells?
1. liposome 2. bilayer lipid membrane
3. squid axon 4. Frank model
3.2. Biophysics of the circulatory system.
Control questions, tasks, assignments for seminars
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The radius of the vessel has halved. How many times will the volumetric blood flow velocity change with a constant pressure drop?
Calculate the blood pressure at a distance of 5 cm from the beginning of the vessel, if at the beginning of the vessel the pressure is 10 4 Pa, its radius is 1 mm, the blood viscosity is 0.005 Pa s, the linear velocity of the blood is 20 cm/s.
How many times will the rate of pressure drop at the beginning of diastole change if the hydraulic resistance of small vessels increased by 20%?
How many times is the hydraulic resistance of an aortic section (aortic radius 1.25 cm) less than the hydraulic resistance of an artery section of the same length (artery radius 2.5 mm)? The viscosity of the blood in the artery is 0.9 of the viscosity of the blood in the aorta.
How many times should the blood pressure increase at the beginning of a large vessel so that when its lumen narrows by 30%, the pressure at the outlet of the vessel and the volumetric blood flow rate remain the same? In the absence of constriction, the pressure drop in the vessel is 0.2 of the pressure at the beginning of the vessel.
in Biology, Candidate of Pediatric Sciences, Associate Professor Osipova I.V. methodical instructions to the student on studying disciplinesDiscipline"Methodology of extracurricular ...
1.Experiments of Pfefer, Hardy-Fischer, Overton. The nature of the cell membrane and the alternative to the cell membrane.
2. The method of fluorescent probes in the study of cell membranes.
3. The specific electrical capacitance of the axon membrane, measured by an intracellular electrode, was 0.5 μF/cm 2 . using the flat capacitor formula, determine the thickness of the hydrophobic layer of the membrane. Ε of lipids is considered equal to 2.
4.Mechanism of generation of action potential of cardiomycetes.
5. Method of spin probes in the study of cell membranes.
6. What is the distance on the surface of the erythrocyte membrane that a phospholipid molecule travels in 1 second as a result of lateral diffusion? The coefficient of lateral diffusion is taken equal to 10 -12 m 2 /s. compare with the circumference of an erythrocyte with a diameter of 8 µm.
7. Structure of the ion channel.
8.Method of differential microcalorimetry.
9. During the phase transition of membrane phospholipids from a liquid-crystal state to a gel, the thickness of the bilayer changes. How will the capacitance of the membrane change in this case?
10. Ion channels of cell membranes.
11. X-ray structural analysis in the study of cell membranes. Principles and examples.
12. During the phase transition of membrane phospholipids from the liquid-crystal state to the gel, the thickness of the bilayer changes. How will the electric field strength in the membrane change in this case?
13. Ionic currents in the axon. Hodgkin-Huxley model.
14.Methods for studying membrane permeability.
15. With the help of spin-labeled phospholipid molecules, the thickness gradient of viscosity in the membrane was established. Describe the experiment.
16.Mechanism of action potential generation.
17. Application of conductometry in the study of membranes. Fricke's experiments.
18. Where the viscosity of the hydrophobic layer is higher: at the membrane surface or in its thickness. How is it installed?
19. Distribution of a nerve impulse along an excitable fiber.
20. Electrokinetic phenomena in cells and suspensions.
21. How will the facilitated diffusion of potassium ions with the participation of the valinomycin molecule change after the phase transition of membrane lipids from a liquid-crystalline state to a gel?
22. Action potential. physical mechanism.
23. Electrosmosis in living cells and tissues.
24. Will there be an osmotic effect (swelling in hypotonic and wrinkling in hypertonic solutions) during the accumulation of sodium ions according to the antiport scheme?
25. Resting potential. His nature.
26. The nature of osmosis in living cells.
27. Will there be an osmotic effect (swelling in hypotonic and wrinkling in hypertonic solutions) during the accumulation of sodium ions according to the symport scheme?
28. Nature of bioelectric potentials.
29. A cell is like an osmometer. An example of determining the isotonicity of a solution using live cells.
30. Show that the Nernst-Planck equation is reduced to the Fick equation for the case of diffusion of uncharged particles.
31. Differences between protein channels and lipid pores.
32. Nature of dead cells settling. Physical chemical basis of the ESR method.
33. The enzyme Na + -K + - ATPase in the plasma membrane of the erythrocyte has completed six cycles. How many sodium and potassium ions were actively transported? How much energy was spent in this case, if the hydrolysis of one mole of ATP is accompanied by the release of 33.6 kJ?. The coupling efficiency is assumed to be 100%.
34.mechanism of membrane permeability for water molecules. The kink hypothesis.
35. NMR spectroscopy in the study of membranes. Examples and principles.
36. Three ion pumps are known in cell membranes: sodium-potassium, proton, and calcium. How is the active transport of sugar and amino acids carried out?
37. Model of pore formation during phase transition.
38.Methods for measuring microviscosity in membranes.
39. Is simultaneous transmembrane transfer of potassium and sodium ions possible according to the symport scheme?
40. electrical breakdown of membrane lipids.
42.methods of spectral probes.
43. Is simultaneous transmembrane transfer of potassium and sodium ions possible according to the antiport scheme?
44. model of the critical lipid pore.
45. application of ion-selective electrodes in the study of membrane permeability.
46. Is simultaneous transmembrane transfer of potassium and sodium ions possible according to the uniport scheme?
47. lipid pores in the light of membrane stability.
48. methods of erythrograms. their informational value.
49. What transport of ions creates a membrane potential difference: passive or active?
50. Mechanism and patterns of secondary active transport of ions.
51. Experimental criteria for facilitated diffusion.
52. What is more the speed of propagation of an electrical signal along the wires of a marine telegraph or the speed of propagation of a nerve impulse along the axon membrane? Why?
53. Electrogenic ion pumps.
54. Cell fractionation methods.
55. What is the biophysical mechanism of action of the local anesthetic tetraylammonium?
56. Ussing's experience and scheme.
57. The nature of the forces of lipid-lipid interaction in the membrane. Research methods.
Calendar thematic plan for the discipline
"Molecular organization of biological membranes"
2011/2012 academic year year (4th year, 7th semester of the All-Russian Biophysics Facility)
the date No. p / p Type and name of the training module Educational and methodological support of the training module LECTURES: Biological membranes as universal structural and functional formations of living systems. Lecture summary. Structural organization of biomembranes. Lecture summary. Membrane proteins and lipids. Lecture summary. Protein-lipid interactions. Lecture summary. Dynamic properties of membranes. Lecture summary. Modeling the structure of membranes. Lecture summary. Membrane structure calculations Lecture summary. TOTAL - 14 hours Workshops * Calculations of electrical capacitance and impedance of membranes. Computer class of the department. Determination of the thickness of the erythrocyte membrane by electrical conductivity. Computer class of the department. Study of the mechanical strength of erythrocyte membranes. Computer class of the department. Study of the effect of cholesterol on the deformability of erythrocyte membranes. Computer class of the department. Calculations of the strength of erythrocyte membranes. Computer class of the department. Study of the effect of a magnetic field on the mechanical properties of erythrocyte membranes Computer class of the department. Total - 22 hours * - each practical lesson is designed for 4 hours.
Approved at a meeting of the department _____________________________________________
1. From this it was concluded that the erythrocyte membrane consists of lipid molecules arranged in two layers.
Apparently, this conclusion of Gorter and Grendel turned out to be correct only due to the mutual compensation of errors, however, in historical terms, this work was of great importance, since since then the concept of the lipid bilayer as the structural basis of biological membranes has become dominant and in fact turned out to be correct.
The concept of a bimolecular lipid membrane was further developed in the 1935 Devson-Danielli model, or "sandwich" model, in which proteins were assumed to cover the surface of the lipid bilayer. This was an unusually successful model, and over the next 30 years, numerous experimental data, especially those obtained using X-ray diffraction and electron microscopy, fully confirmed its adequacy. However, at the same time, it was discovered that membranes perform a huge variety of functions, and in order to explain this phenomenon, the original Devson-Danielli model was repeatedly modified.
The rapid progress in membranology, which has resulted in the formation of modern concepts, has been achieved largely due to advances in the study of the properties of membrane proteins. Electron microscopic studies using the freeze-shear method showed that globular particles are embedded in the membranes. Meanwhile, biochemists using detergents managed to dissociate membranes to the state of functionally active "particles". Spectral data indicated that membrane proteins are characterized by a high content of a-helices and that they probably form globules rather than being distributed as a monolayer on the surface of the lipid bilayer. The nonpolar properties of membrane proteins suggested the presence of hydrophobic contacts between the proteins and the inner nonpolar region of the lipid bilayer. At the same time, methods were developed that made it possible to reveal the fluidity of the lipid bilayer. Singer and Nicholson brought all these ideas together to create a fluid mosaic model. Within this model, the membrane is represented as a fluid phospholipid bilayer, in which freely diffusing proteins are immersed. The old Devson-Danielli model was static and successfully explained the structural data available at the time, obtained at a fairly low resolution. At the same time, since 1970, much attention has been paid to the study of dynamic properties and their relationship with membrane functions. In recent years, the fluid mosaic model has also been modified, and this process will continue. In particular, it has now become clear that not all membrane proteins diffuse freely in the liquid lipid bilayer. There are data on the existence of lateral j-domains in the membrane itself. The role of the cytoskeleton is also being carefully studied. It is becoming increasingly clear that some portions of the membranes appear to differ in structure from the classical lipid bilayer. Nevertheless, in the foreseeable future, the fluid mosaic model in its various modifications will serve as a conceptual basis for many membrane studies.
3. Morphology of membranesTwo methods played an important role in elucidating the morphology of membranes: x-ray diffraction and electron microscopy. It was with their help that the correctness of the bilayer model was confirmed. However, it should be borne in mind that both of these methods face a number of limitations in elucidating a detailed picture of the molecular organization of membranes.
3.1 X-ray diffraction
In the study of highly ordered crystalline samples using the X-ray diffraction method, it is possible to obtain information about the structure with high resolution. In the case of poorly ordered preparations, the possibilities of this method are limited. Some specialized membrane systems already have a regular structure, and therefore they can be studied by X-ray diffraction methods. An example of this kind is the myelin sheath of peripheral nerve fibers; it is a membrane that, repeatedly wrapping around the axon, forms a regular system of concentric membrane structures. X-ray diffraction studies on myelin, carried out back in the 1930s, confirm the adequacy of the bilayer model of membranes. The same conclusion is drawn by the study of the outer segment of rods of the retina of vertebrates, which are natural ordered membrane systems, as well as artificially ordered systems that are formed during the collapse under centrifugation conditions of membrane vesicles obtained from mitochondria and erythrocytes. In all these cases, a similar distribution of electron density in the membrane was observed, shown in Fig. 1.4
To interpret X-ray diffraction data, it is necessary to determine not only the intensities of reflections, but also their phases. In the case of regularly packed membrane systems, the problem is greatly simplified, since these systems consist of repeating elements with central symmetry.
The obtained data show that the structure of all membranes is similar: they have a hydrophobic inner region with a low electron density and two layers of polar groups with a high electron density. The X-ray diffraction data obtained for different membranes differ only slightly, despite the large differences in their protein content. Although X-ray diffraction data provide some information about how the bulk of membrane proteins are located in the membrane, in general, the X-ray diffraction analysis method does not provide a detailed molecular picture.
Wilkins et al. noted in 1971 that X-ray diffraction can also be used to study aqueous dispersions of membranes and phospholipids. In this case, the reflections generated by the polar regions on both sides of the bilayer make it possible to find its thickness equal to the distance between the polar heads, and the distance between these chains can be determined from the reflections generated by ordered hydrocarbon chains. In this case, too, membrane preparations obtained from different sources gave a similar diffraction pattern, which confirms the universality of the bilayer model.
The impossibility of obtaining a detailed molecular pattern using the diffraction method limits the application of this method to the study of biological membranes. However, it can be very useful in the study of ordered lipid-aqueous systems.
3.2 Electron microscopy
Transmission electron microscopy of thin sections of myelin, and in fact of all other membranes, reveals a characteristic three-layer structure consisting of two electron-dense bands separated by a gap of about 80 A. This picture is obtained largely as a result of the treatment of preparations with osmium tetroxide, usually used in this method. Robertson called the observed structure "unitary" to emphasize its universality, and although the molecular mechanisms of membrane staining with osmium are unknown, this structure was considered as confirmation of the validity of the bilayer model of the membrane. It is clear, however, that membranes may be adversely affected during the preparation of specimens for transmission electron microscopy. In particular, it is known that treatment with osmium tetroxide leads to a significant loss of protein from the erythrocyte membrane. And although the three-layer structure observed in this case reflects to some extent the organization of bilayer membranes, more detailed information on protein localization cannot be obtained by this method.
Some information about the arrangement of membrane proteins was provided by new methods, which have now become "classical" - the methods of freezing-cleavage and freezing-etching. In these cases, the preparations are quickly frozen without exposing them to any damaging effects, as when obtaining thin sections. The drug preparation process includes the following operations.
After freezing, the sample, which is a suspension of cells or membranes, is cut off with a knife at low temperature in a high vacuum. The forces generated during chipping lead to the formation of a cut that passes through the sample. It turned out that when the cut plane passes through the membrane, the latter splits mainly along its middle region and splits into two halves. As a result, the internal region of the membrane is exposed on the formed cleavage planes.
If necessary, the sample is subjected to etching - the usual sublimation of ice is carried out in a vacuum. This allows better visualization of the surface structures of cell membranes.
After that, a so-called replica from the exposed surface is obtained. It is this replica that is studied under an electron microscope. To obtain a replica, platinum is first deposited on the sample at an angle of about 45° in order to reveal the topological characteristics of the preparation. Then the platinum replica is given mechanical strength by applying a layer of carbon on it. After that, the preparation is thawed, the replica floats up, and it is caught using a special net.
The most characteristic structures observed in the study of membranes by the freeze-cleavage method are numerous intramembrane particles with a diameter of 80 to 100 Å, lying in the plane of membrane cleavages. Usually they are located randomly, but sometimes they form groups. Numerous studies have shown that these particles are possibly membrane proteins. Curiously, electron microscopy of thin sections does not reveal such structures. The replicas obtained from the two halves of the split membrane are not always topologically complementary. This means that some particles are bound to only one of the halves of the membrane. Freeze-cleavage data were widely used by Singer and Nicholson in the development of the fluid mosaic model of membranes, as they convincingly showed that globular proteins are located not only on the surface of the membrane, but also inside the bilayer.
Figure 1.6 shows an electron micrograph of a preparation of proteoliposomes reconstructed from egg phosphatidylcholine and an unfractionated preparation of band 3 protein from a human erythrocyte membrane; The preparation was obtained by the freezing-cleaving method.
The band 3 protein is the main protein component of the erythrocyte membrane and is known to transport anions. If phospholipid vesicles do not contain this protein, then the resulting frozen chip preparations have a smooth surface.
Upon incorporation of the band 3 protein into phospholipid vesicles, intramembrane particles appear on the cleavages, which are practically indistinguishable from the particles observed in erythrocyte membranes. Moreover, at pH 5.5, the particles seen in the erythrocyte membrane aggregate, and this aggregation is carried out as a result of the interaction of the band 3 protein with two other proteins, spectrin and actin.
The latter are components of the cytoskeleton located on the inner surface of the erythrocyte membrane. The reconstructed system consisting of the band 3 protein and phosphatidylcholine behaves similarly, with particle aggregation observed in the presence of spectrin and actin at pH 5.5, but not at pH 7.6.
These data further strengthened the concept of membrane proteins as globular particles freely moving in the membrane plane. Interestingly, static microphotographs of preparations obtained by the freeze-chipping method helped researchers in studying the dynamic properties of membranes. As we will see, there are many proteins in membranes that cannot swim freely in the lipid sea.
4. Isolation of membranesOver the past three decades, it has become increasingly clear that the vast majority of cellular functions are carried out with the direct involvement of membranes.
Both plant and animal cells are divided into compartments, and many cytoplasmic organelles, as shown in Section 1.1, are of a membrane nature.
In addition to the organelles characteristic of most cells, there are also specialized membrane systems, such as the sarcoplasmic reticulum of muscle cells, the myelin sheath of peripheral nerve fibers, the thylakoid membranes of chloroplasts, and the membranes of disks in retinal rods. Prokaryotic organisms also have membranes, although not as developed as eukaryotic ones.
Gram-positive bacteria, such as Bacillus subtilis, have only a cytoplasmic membrane, while Gram-negative bacteria, such as Escherichia coli, also have an outer one located on top of a thin peptidoglycan cell wall.
Some specialized organelles have also been found in prokaryotic cells. Some viruses pathogenic to animals, such as enveloped viruses, have a true membrane, and such membranes have proven to be extremely interesting to study.
The study of membranes, as a rule, is associated with their purification, and each type of membrane is characterized by its own conditions for preparative isolation.
So, if you have to study the plasma membrane of any cells, then you first need to isolate these cells from the tissue. The optimal conditions for disrupting cells and separating the membranes of interest from other cellular components must then be selected. The purity criteria of isolated membranes deserve special attention.
4.1 Destruction of cells
It is desirable to choose a technique that effectively destroys the cells themselves while maintaining the structure of the membranes to be isolated. For many animal cells, a relatively gentle procedure such as homogenization in glass-walled Downs or Potter-Elveheim homogenizers with a Teflon pestle can be used. In this case, the cells are destroyed due to shear forces that occur when the suspension is forced through a narrow gap between the Teflon pestle and the glass wall of the homogenizer. With this treatment, the plasma membrane "breaks down" and the bonds between various organelles are destroyed while maintaining the integrity of the organelles themselves. Using this procedure, specialized regions of the plasma membrane can also be separated from each other, for example, the basolateral or apical regions of the membrane of epithelial cells. It is desirable to operate under conditions where the integrity of the organelles is maintained to minimize the possibility of release of hydrolytic enzymes and to facilitate subsequent membrane separation operations.
For the destruction of cells with a wall, more stringent methods are required. Sometimes, before cells are destroyed, they are first treated with enzymes that break down components of the cell wall to facilitate its subsequent destruction. For example, treatment with Tris-EDTA buffer and lysozyme is used to destroy E. coli cells. More stringent techniques include rubbing the cells, sonicating them, and extruding them. Grinding is usually carried out in the presence of various abrasive materials - sand, alumina or glass beads. Small volumes of material can be ground in a mortar and pestle, but for larger volumes special mechanical devices must be used. Bacterial cells are often destroyed using ultrasound. It is believed that in this case, the destruction occurs under the action of shear forces resulting from cavitation. The same forces occur when a cell suspension is forced through a small hole, for example, when cells are destroyed using a French press. There are many varieties of these methods, and their choice depends on the characteristics of the membrane system that is to be studied.
It should be noted that membrane fragments obtained during cell destruction usually spontaneously form vesicles. An example is:
1) microsomes derived from the plasma membrane, endoplasmic reticulum, or specialized systems such as the sarcoplasmic membrane;
2) submitochondrial particles from the inner mitochondrial membrane;
3) synaptosomes formed when nerve endings are torn off in the area of synaptic contacts;
4) bacterial membrane vesicles formed from the plasma membrane of E. coli. Vesicles are also formed from other membrane systems, for example, from the membranes of the Golgi apparatus. Their size in most cases strongly depends on the method of cell destruction. This is especially important, since the size of vesicles largely determines the rate of their sedimentation during centrifugation and their behavior in the subsequent stages of membrane purification. Some membranes do not form vesicles, in particular the membranes of the lateral surfaces of animal cells in contact with each other. When such cells are destroyed, a pair of adjacent membrane fragments is torn off, held together by the contact area. The presence of such contacts prevents the closure of fragments into vesicles, so the membranes are released in the form of plates or ribbon-like structures.
Of great importance in the destruction of cells is also the correct choice of medium. For example, in order to keep membrane organelles closed, one should use a medium that is isoosmotic to their internal contents. Most often, a sucrose solution is used for this at a concentration of 0.25-0.30 M. In some cases, it is better to use sorbitol and mannitol. It should be noted that the preservation of isotonicity also plays an important role at subsequent stages of the preparative isolation of intact organelles.
4.2 Separation of membranes
Currently, centrifugation is most commonly used to separate membranes. Membrane particles can be classified according to their sedimentation rate or buoyant density. The first method is called zonal centrifugation and separation occurs according to S values, and the second method is isopycnic centrifugation and separation occurs under density equilibrium conditions. In practice, some hybrid of these two methods is usually used. Figure 1.7 shows the position of some subcellular units on the "S-g" coordinate plane.
The abscissa shows the particle sedimentation coefficients, and the ordinate shows the density.
The principle of separation by sedimentation rate can be easily understood by comparing the S values for different fractions. For example, nuclei have relatively high S values, i.e. their sedimentation rate is much higher than that of most other subcellular organelles. Nuclei can be selectively pelleted by centrifugation of the cell homogenate, leaving all other organelles in the supernatant. At the same time, smooth and rough endoplasmic reticulum cannot be separated using zonal centrifugation.
Differences in their density are often used to isolate different membrane fractions from a cell homogenate. For this purpose, centrifugation in a density gradient is carried out. Most often, sucrose is used to create a density gradient, but this method has serious drawbacks. To obtain the density required to separate the different membrane fractions, it is necessary to prepare solutions with a high concentration of sucrose, which have a high viscosity and are also hypertonic. The introduction of subcellular organelles into a hypertonic sucrose solution leads to their dehydration, and the subsequent adjustment of the solution to isotonic conditions is often accompanied by lysis and damage to the organelles. Another problem is that many membrane organelles are permeable to sucrose. It can also lead to osmotic destruction of organelles. Penetration of sucrose into separable membrane organelles can change their effective density.
Table 1.1. Physical time is increasingly using other media to create a density gradient. Some of these environments are listed in Table 1.1
To solve these problems, the last properties of gradient media.
1. Ficoll. A high molecular weight hydrophilic polymer of sucrose that can be used to prepare solutions of C "Density up to 1.2 g / ml. Its main advantage is the low osmotic pressure of solutions compared to solutions with an equivalent concentration of sucrose. Due to this, it is possible to create solutions that are isotonic throughout range of concentrations due to the additional inclusion of sucrose or physiologically acceptable salts in the medium.The disadvantages are the high viscosity of the resulting solutions and the significantly non-linear dependence of viscosity and osmolarity on concentration.
2. Metrizamide. Triiodosubstituted glucose benzamide Metrizamide solutions have a higher density than ficoll solutions at the same concentrations. The main advantage of metrizamide solutions is their very low viscosity, which allows faster separation. The 35% metrizamide solution has an almost physiological osmolarity, so that most operations during membrane separation can be carried out without exposing them to hypertonic solutions. Sodium metrizoate is a related compound with similar properties to metrizamide, with the only difference that its solution is isotonic at a concentration of about 20%. Sodium metrizoate is used primarily for the isolation of intact cells. Naikodenz is also a derivative of triiodobenzoic acid, but has three hydrophilic side chains. When centrifuged, it rapidly develops its own density gradient; used to isolate subcellular organelles.
Percoll. Colloidal suspension of silica gel coated with polyvinylpyrrolidone. This coating reduces the toxic effect of silica gel. The main advantage of percoll is that it does not penetrate biological membranes, and its solutions have low viscosity and low osmolarity. Due to the large particle size, centrifugation of the Percoll solution at moderate speeds results in the formation of a density gradient. Therefore, separation usually occurs very quickly. The medium used for centrifugation may be isotonic throughout the volume due to the inclusion of salts or sucrose in it. It is not difficult to create a gentle gradient, which makes it possible to carry out a very efficient separation of membrane fractions according to their buoyant density.
Sorbitol and mannitol. These substances are sometimes used instead of sucrose because, according to published data, they penetrate through some biological membranes worse than sucrose.
Note that glycerol is not used to create a density gradient because it cannot achieve sufficiently high density values. Alkali metal salts such as CsCl are used only when high density solutions are required. However, it should be borne in mind that at the concentrations required to create an equilibrium density, these salts often have a damaging effect on membrane organelles.
Other methods are also used to isolate membranes from cell homogenates, although not as frequently as centrifugation.
1. Phase distribution. In this case, the separation of membrane particles occurs in accordance with their surface properties. For this purpose, two immiscible layers of aqueous solutions of various water-soluble polymers are formed. Examples are mixtures of polyethylene glycol dextran and dextranficoll. Membrane particles are separated according to their affinity for these phases. The latter can be selected so as to separate the membranes by their surface charge or hydrophobicity.
Continuous free flow electrophoresis. In this case, the separation of particles occurs in accordance with their electric charge. The drug to be divided is continuously introduced into a thin layer of buffer flowing down a vertical wall. In this case, an electric field is applied perpendicular to the flow direction. Thus, the electrophoretic separation of the particles occurs across the flowing buffer, which is collected at the bottom of the chamber in the form of separate fractions.
affinity adsorption. The separation is based on a biospecific interaction between the membrane components and the solid phase. With the discovery of monoclonal antibodies, it became possible to create preparative techniques based on the use of specific antigenic components for membrane isolation. The resulting antibodies can be covalently attached to a solid support and with their help to carry out the specific binding of the respective membranes. Most often, this method is used to isolate membrane proteins. One of the problems that arise here is related to the selection of membrane elution conditions that would not cause protein denaturation.
A method based on the use of silica gel microgranules. Usually, the share of plasma membranes accounts for no more than 1°7o of the total mass of all membranes of eukaryotic cells. Therefore, the isolation of absolutely pure plasma membranes is associated with great difficulties. One approach that has been developed specifically for the isolation of plasma membranes is based on the use of cationized silica gel microbeads. These granules are strongly adsorbed on the outer surface of the plasma membrane of intact cells, and the fraction of plasma membranes associated with the granules is easily separated in the sucrose density gradient from other membranes due to the higher density of the granules. A feature of this method is that in the resulting preparation, the plasma membrane with its inner surface is turned into a solution.
4.3 Purity criteria for membrane fractions
Perhaps the most objective criterion for the purity of the isolated membrane fraction is the presence in it of some unique component that is contained only in this membrane or is predominant in it. Typically, such components are enzymes, which in this case are called markers. The list of marker enzymes that are used to control the purity of membrane fractions is given in Table 1.2. When determining the activity of an enzyme, it should be taken into account that it can be in a latent form, for example, due to the fact that it is localized on the inner surface of the secreted membrane vesicles. Other problems associated with the evaluation of the purity of isolated membranes are considered in the review. It should be noted that the recommended methods in most cases are well developed and standardized.
In some cases, more convenient membrane markers are not enzymes, but specific receptors for lectins, hormones, toxins, or antibodies. If the systems under study are well characterized, then the purity of the membrane fraction can be judged by its protein composition determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. For example, the outer membrane of Gram-negative bacteria has a characteristic set of polypeptides that are not present in the cytoplasmic membrane.
Table 1.2 Markers used to control the purity of membrane fractions isolated from mammalian cells "
Membrane fraction marker enzyme Plasma membranes 5"-Nucleotidase Alkaline phosphodiesterase Na * / K + -ATPase (basolateral-
epithelial membrane cells) Adenylate cyclase (basal hepatocyte membrane) Aminopeptidase (membrane brush border epithelium) Mitochondria (internal Cytochrome c oxidase membrane) Succinate-cytochrome c-oxido- reductase Mitochondria (outer Monoamine oxidase membrane) Lysosomes Acid phosphatase 0-Galactosendase Peroxisomes Catalase urate oxidase D-amino acid oxidase Apparatus membranes Galactosyltransferase Golgi Endoplasmic Glucose-6-phosphatase reticulum Choline phosphotransferase NADPH-cytochrome c-oxido- reductase Cytosol lactate dehydrogenase Other criteria that can be used to judge the purity of membranes include their morphology, which is detected using electron microscopy, and the characteristics of the chemical composition. For example, fractions representing the plasma membrane, the Golgi apparatus, or mitochondria can be identified by their morphology. In some cases, the drug is characterized by the content of cholesterol in it. For example, mitochondrial membranes contain much less cholesterol than Golgi and plasma membranes.
Detergent molecules per micelle. In membrane research, a rather limited range of detergents is used. In table. 1 presents those that are most often used for solubilization and reconstruction of membranes. These detergents are characterized by rather high CMC values (10-4-10-2 M) and the fact that they belong to the category of so-called soft detergents, that is, such ...
Bilayer formation is a special property of lipid molecules and is realized even outside the cell. The most important properties of the bilayer: - ability to self-assembly - fluidity - asymmetry. 1.2. Although the main properties of biological membranes are determined by the properties of the lipid bilayer, most of the specific functions are provided by membrane proteins. Most of them penetrate the bilayer in the form of a single...
The study of proteins contained in the plasma membrane of erythrocytes made it possible to formulate new ideas about the structure of membranes. In particular, there was an assumption that at least some membranes have a "skeleton". The human erythrocyte membrane contains five major proteins and a large number of minor ones. Most membrane proteins are glycoproteins. The integral proteins in the erythrocyte membrane include glycophorin (“sugar carrier”). Its molecular weight is 30,000; glycophorin contains 130 amino acid residues and many sugar residues, which account for about 60% of the entire molecule. At one end of the polypeptide chain is a hydrophilic head of complex structure, which includes up to 15 oligosaccharide chains, each of which consists of approximately 10 sugar residues. At the other end of the polypeptide chain of glycophorin is a large number of residues of glutamic and aspartic acids (Fig. 12-20), which at pH 7.0 carry a negative charge. In the middle of the molecule, between the two hydrophilic ends, there is a section of the polypeptide chain containing about 30 hydrophobic amino acid residues. The sugar-rich end of the glycophorin molecule is localized on the outer surface of the erythrocyte membrane, protruding from it in the form of a bush. It is believed that the hydrophobic region located in the middle of the glycophorin molecule passes through the lipid bilayer, and the polar end with negatively charged amino acid residues is immersed in the cytosol. The sugar-rich head of glycophorin contains antigenic determinants that determine the blood type (A, B, or O). In addition, it has sites that bind some pathogenic viruses.
The share of another important protein of the erythrocyte membrane - spectrino - accounts for up to 20% of the total amount of proteins in the membrane.
Rice. 12-20. Glycophorin molecule in the erythrocyte membrane. Branched carbohydrate chains protruding from the membrane carry specific sites that determine the blood group, as well as sites responsible for the binding of certain viruses.
This peripheral protein is located on the inner surface of the membrane; it is easy to extract. The spectrin molecule consists of four polypeptide chains, the total molecular weight of which is about 1 million; these chains form long flexible rods 100–200 nm long. By binding to certain proteins and lipids on the inner surface of the erythrocyte membrane, spectrin molecules form a flexible lattice, which, apparently, plays the role of the membrane skeleton. Actin microfilaments also bind to spectrin, and it is very likely that they connect the spectrin rods to each other. Thus, we can say that the erythrocyte membrane has a skeleton, or framework, on which specific lipids and membrane proteins are attached (Fig. 12-21).
Plasma membranes of other cells have a more complex structure.
Rice. 12-21. Schematic representation of a section of the erythrocyte membrane. The scheme shows oligosaccharide "antennas" formed by membrane glycoproteins and glycolipids, side oligosaccharide chains of glycophorin, as well as a skeletal base of spectrin molecules attached to the inner surface of the membrane, interconnected by short actin filaments.
On the outer surface of cells in many dense tissues, there is another important glycoprotein, fibronectin (Sec. 11.12), which has a high adhesive ability and, possibly, provides adhesion of cells of the same type to each other.
in the opposite direction
