EntranceActin and myosin perform a transport function. Contractile proteins
The protein composition of muscle tissue is very complex. It has been studied by many scientists for a long time. The founder of Russian biochemistry, A. Ya. Danilevsky, studying the proteins of muscle tissue, gave a correct idea of the physiological role of a number of proteins and the significance of the contractile protein myosin contained in myofibrils.
Subsequently, myosin was studied by V. A. Engelhardt, I. I. Ivanov and other Soviet scientists. The Hungarian scientist Szent-Georgyi made a great contribution to the study of muscle contraction. Another Hungarian scientist, Straub, discovered the muscle protein actin.
The study of muscle tissue should begin with proteins, since they account for about 80% of the dry residue of muscle tissue. In accordance with the morphological structure of the muscle fiber, proteins are distributed as follows:
From the above diagram it can be seen that the protein composition of muscle tissue is very diverse. Sarcoplasm contains four proteins: myogen, myoalbumin, globulin X and myoglobin. Myofibrils contain a complex of actin and myosin called actomyosin. All sarcoplasmic proteins are called intracellular, and sarcolemma proteins are called extracellular. The nuclei contain nucleoproteins, and the sarcolemma contains collagen and elastin. If we consider that muscle tissue, in addition, contains a significant amount of different enzymes and each of them is a special protein, then the protein composition of muscle tissue turns out to be even more complex.
Myosin
The main protein of muscle tissue is myosin. It makes up almost half of all muscle tissue proteins, and it is found in the muscles of all mammals, birds and fish. In terms of nutritional value, it is a complete protein. In table Figure 7 shows the amino acid composition of bovine myosin.
Myosin was studied in detail by Soviet biochemists, who discovered that it is not only structural protein muscle tissue, i.e. a protein involved in the construction of the cell, but also an enzyme - adenosine triphosphatase, which catalyzes the ATP hydrolysis reaction. In this case, ADP (adenosine diphosphoric acid) and phosphoric acid are formed and a large amount of energy is released, which is used in muscle work.
Myosin was obtained in pure crystalline form. Its molecular weight is very large, approximately 1.5 million. Crystalline myosin, in the complete absence of salts, is perfectly soluble in water. But it is enough to add an insignificant amount of any salt, for example sodium chloride, to water, and it completely loses its ability to dissolve and dissolution occurs already at a sodium chloride concentration of about 1%. However, in relation to salts, for example ammonium sulfate, myosin behaves like a typical globulin.
When meat proteins are extracted with water, myosin does not go into solution. When processing meat with saline solutions, it is found in the salt extract. When a saline solution of myosin is diluted with water, the salt concentration decreases and myosin begins to precipitate. Myosin is salted out when completely saturated with sodium chloride and magnesium sulfate (salting out is done with crystalline salt, otherwise it is impossible to achieve complete saturation).
The isoelectric point of myosin is at pH 5.4-5.5.
Myosin has the property of entering into special bonds with various substances, primarily proteins, to form complexes. A special role in muscle activity is played by the complex of myosin and actin - actomyosin.
Actin and actomyosin
The actin protein can exist in two forms: fibrillar and globular. In resting muscle, actin is in fibrillar form; with muscle contraction it becomes globular. Great importance adenosine triphosphoric acid and salts have in this transformation.
Muscle tissue contains 12-15% actin. It goes into solution during prolonged extraction with saline solutions; with short-term extraction it remains in the stroma. The molecular weight of actin is about 75,000.
When solutions of actin and myosin are mixed, a complex called actomyosin is formed, from which myofibrils are mainly built. This complex is characterized by high viscosity and is capable of contracting sharply at certain concentrations of potassium and magnesium ions (0.05 m KCl > and 0.001 m MgCl2) in the presence of adenosine triphosphate. At higher salt concentrations (0.6 m KCl), actomyosin breaks down into actin and myosin when ATP is added. The viscosity of the solution decreases noticeably.
According to Szent-Georgia, compression of actomyosin under the influence of ATP underlies the contraction of living muscle.
Actomyosin, like a genuine globulin, is insoluble in water. When meat is processed with saline solutions, actomyosin with an uncertain actin content passes into the solution, depending on the duration of extraction.
Globulin X
Muscle tissue contains about 20% globulin X of the total protein. It is a typical globulin, that is, it does not dissolve in water, but dissolves in saline solutions of average concentration; precipitates from solutions at half saturation with ammonium sulfate (1 volume of protein solution and 1 volume of saturated ammonium sulfate solution), with sodium chloride at full saturation.
Miogen
Muscle tissue contains about 20% myogen of the total protein. It cannot be classified as a typical albumin or globulin, since it dissolves in water, is not sufficiently salted out with sodium chloride and magnesium sulfate upon saturation (crystalline salt), while at the same time it is precipitated with ammonium sulfate at 2/3 of saturation (1 volume of protein solution and 2 volumes saturated solution of ammonium sulfate). This protein was obtained in crystalline form. The molecular weight of myogen is 150,000.
V. A. Engelhardt discovered in myogen the ability to catalyze one of the most important reactions occurring in the process of glycolysis of muscle tissue. This discovery was the first to show that structural proteins, i.e., proteins involved in the construction of tissues, can have enzymatic activity.
Myoalbumin
Muscle tissue contains about 1-2% myoalbumin of the total protein. It is a typical albumin, i.e. it dissolves in water, is not precipitated by sodium chloride upon saturation, but is precipitated by ammonium sulfate.
Myoglobin
Myoglobin is a complex chromoprotein protein with a molecular weight of 16,900. During hydrolysis, it breaks down into the globin protein and the non-protein heme group. Myoglobin colors muscles red; It differs from hemoglobin in its protein part; their prosthetic group is the same.
Upon oxidation, heme transforms into hematin, and in the presence of hydrochloric acid- in hemin. The content of hemin can be used to judge the amount of myoglobin in muscle tissue.
The hemin content in cattle muscles ranges from 42 to 60 mg per 100 g of tissue; in the muscles of pigs it is much less - from 22 to 42 mg per 100 g of tissue, so they are less colored.
Myoglobin, like blood pigments, have a characteristic absorption spectrum.
The principle of obtaining absorption spectra of colored substances, in particular meat and blood pigments, is that light energy passing through a pigment solution is absorbed by this solution. In this case, the so-called absorption (absorption) of light occurs, which can be detected with a spectroscope.
The characteristic absorption bands for muscle tissue and blood pigments range from 400 to 700 mm. In this interval, waves are perceived by our eye, and we can see dark bands in the spectrum using a spectroscope, resulting from the absorption of light with a certain wavelength.
The absorption of light by colored substances can be quantified using a spectrophotometer. The results obtained are usually expressed graphically. In this case, the wavelength of light is plotted along the abscissa axis, and the percentage of light passed through the solution is plotted along the ordinate axis. The less light passed, the more of it was absorbed by the colored substance. The total transmission of light by the solution is taken as 100%.
In Fig. Figure 10 shows the absorption (absorption) of light by a solution of oxymyoglobin; It shows that oxymyoglobin has two pronounced characteristic absorption bands in the visible region of the spectrum, i.e., two regions in which it transmits the least light and, therefore, absorbs the most light. The maxima of these sections are at two wavelengths; λ 585 mmk and λ 545 mmk,
In Fig. Figure 11 shows a spectrophotometric curve of oxyhemoglobin for comparison.
Myoglobin has a greater ability to bind to oxygen than blood hemoglobin. Through myoglobin, muscle tissue is supplied with oxygen. Working muscles contain more myoglobin, since oxidation occurs more intensely in them. It is known that the muscles of the legs are more strongly colored than the back muscles; the muscles of working oxen are also more colored than those of non-working animals. This is especially noticeable in birds, whose pectoral muscles, being non-working, are almost not colored.
Collagen and elastin
Collagen and elastin are connective tissue proteins that are insoluble in water and saline solutions. They form the sarcolemma - the thinnest sheath of muscle fiber.
Nucleoproteins
Nucleoproteins are proteins that make up the cell nucleus. Characteristic feature their ability to dissolve in solutions of weak alkalis. This is explained by the fact that their molecule contains a prosthetic group that has acidic properties.
Separation of muscle proteins
When muscle tissue is treated with saline solutions of medium concentration, its proteins can be divided into stromal proteins and plasma proteins. The stroma refers to the saline-insoluble structural basis of muscle tissue, which consists mainly of sarcolemmal proteins (see diagram).
The solubility of intracellular proteins in muscle tissue varies. For example, actomyosin and globulin X are insoluble in water and are more easily precipitated from saline solutions by ammonium sulfate and sodium chloride than myogen. Myogen dissolves in water like myoalbumin, but differs from it in its salting properties.
The solubility of muscle tissue proteins in salt solutions at a neutral reaction and their precipitation are given in Table. 8.
When salting, cooking and other types of technological processing of meat, protein substances are lost. The magnitude of protein losses is due to their different solubility and sedimentability.
Knowing the properties of proteins, it is possible to select conditions under which losses will be minimal. Therefore, special attention should be paid to the study of these properties of proteins.
Structural function of proteins
Structural function of proteins is that proteins
- participate in the formation of almost all cell organelles, largely determining their structure (shape);
- form the cytoskeleton, which gives shape to cells and many organelles and provides mechanical shape to a number of tissues;
- are part of the intercellular substance, which largely determines the structure of tissues and the shape of the body of animals.
Proteins of the intercellular substance
In the human body there are more intercellular proteins than all other proteins. The main structural proteins of the intercellular substance are fibrillar proteins.
Collagens
Collagens are a family of proteins; in the human body they constitute up to 25 - 30% of the total mass of all proteins. In addition to the structural function, collagen also performs mechanical, protective, nutritional and reparative functions.
The collagen molecule is a right-handed helix of three α-chains.
In total, humans have 28 types of collagen. They are all similar in structure.
Elastin
Elastin is widely distributed in connective tissue, especially in the skin, lungs and blood vessels. General characteristics for elastin and collagen are high in glycine and proline. Elastin contains significantly more valine and alanine and less glutamic acid and arginine than collagen. Elastin contains desmosine and isodesmosine. these compounds can only be found in elastin. Elastin is insoluble in aqueous solutions(like collagen), in solutions of salts, acids and alkalis, even when heated. Elastin contains a large number of amino acid residues with non-polar side groups, which apparently determines the high elasticity of its fibers.
Other extracellular matrix proteins
Keratins are divided into two groups: α-keratins and β-keratins. The strength of keratin is perhaps second only to chitin. A characteristic feature of keratins is their complete insolubility in water at pH 7.0. They contain the residues of all amino acids in the molecule. They differ from other fibrillar structural proteins (for example, collagen) primarily in the increased content of cysteine residues. The primary structure of the polypeptide chains of a-keratins has no periodicity.
Other intermediate filament proteins
In other types of tissues (except epithelia), intermediate filaments are formed by proteins similar in structure to keratin - vimentin, neurofilament proteins, etc. Lamin proteins in most eukaryotic cells form the inner lining of the nuclear membrane. The nuclear lamina, which consists of them, supports the nuclear membrane and is in contact with chromatin and nuclear RNAs.
Tubulin
Structural proteins of organelles
Proteins create and determine the shape (structure) of many cellular organelles. Organelles such as ribosomes, proteasomes, nuclear pores, etc. consist mainly of proteins. Histones are necessary for the assembly and packaging of DNA strands into chromosomes. The cell walls of some protists (for example, Chlamydomonas) consist of proteins; The cell wall of many bacteria and archaea contains a protein layer (S-layer), which is attached to the cell wall in gram-positive species, and to the outer membrane in gram-negative species. Prokaryotic flagella are made from the protein flagellin.
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ACTIN
one of the main proteins will be reduced. muscle fiber elements. It can exist in the form of a monomer (G-A., mol. wt. approx. 42 thousand) and in polymerization. condition (F-A.).
Molecule G-A. has a globular two-domain form and is associated with one ATP molecule, which is converted into adenosine diphosphate during the polymerization of G-A. In salt-free water solutions G-A. did not polymerize. In the case of adding KS1 or MgCl 2, the process begins at a concentration of resp. 0.1-0.15 or 0.01 M. Possibility of polymerization of G-A. in the body depends on actin-binding proteins, for example. filamin, actinin.
FA is a linear polymer that forms a flat helix (its threads are polar) with a pitch of 38 nm and a subunit diameter of 5.5 nm. One turn of the spiral contains 13-14 molecules G-A. Polymerization of the monomer leads to a sharp increase in the viscosity of the solution. F. forms a complex with others. protein - myosin - and has a strong activating effect on its adenosine triphosphatase. An important property of FA is the ability to coordinate metabolic processes, which manifests itself during its interaction. with a number of enzymes (phosphorylase kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, etc.).
A. is present in all eukaryotic cells (10-15% by weight of all proteins). In non-muscle cells, it forms the “cytoskeleton” (microfilaments of the cell cytoplasm).
Lit.: Fundamentals of biochemistry, trans. from English, vol. 3, M., 1981, p. 1406-10. B. F. Poglazov.
Chemical encyclopedia. - M.: Soviet Encyclopedia. Ed. I. L. Knunyants. 1988 .
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Actin actin. Muscle fiber protein (molecular weight 42 kDa), exists in two forms, fibrillar and globular, has sections complementary to sections of myosin molecules
Muscle contraction is based on the mutual movement of two systems of filaments formed by actin and myosin. ATP is hydrolyzed at the active site located in the myosin heads. Hydrolysis is accompanied by a change in the orientation of myosin heads and movement of actin filaments. Regulation of contraction is provided by special Ca-binding proteins located on actin or myosin filaments.
Introduction. Various shapes motility is characteristic of almost all living organisms. During evolution, animals have developed special cells and tissues whose main function is to generate movement. Muscles are highly specialized organs capable of generating mechanical forces through the hydrolysis of ATP and ensuring the movement of animals in space. At the same time, the contraction of muscles of almost all types is based on the movement of two systems of protein threads (filaments), built mainly from actin and myosin.
Ultrastructure of muscles. For highly efficient conversion of ATP energy into mechanical work, muscles must have a strictly ordered structure. Indeed, the packing of contractile proteins in a muscle is comparable to the packing of atoms and molecules in a crystal. Let's look at the structure of skeletal muscle (Fig. 1).
The fusiform muscle consists of bundles of muscle fibers. Mature muscle fiber is almost completely filled with myofibrils - cylindrical formations formed from a system of overlapping thick and thin filaments formed by contractile proteins. In skeletal muscle myofibrils, a regular alternation of lighter and darker areas is observed. Therefore, skeletal muscles are often called striated. The myofibril consists of identical repeating elements, the so-called sarcomeres (see Fig. 1). The sarcomere is bounded on both sides by Z-discs. Thin actin filaments are attached to these discs on both sides. Actin filaments have a low density and therefore appear more transparent or lighter under a microscope. These transparent, light areas located on both sides of the Z-disk are called isotropic zones (or I-zones) (see Fig. 1). In the middle of the sarcomere there is a system of thick filaments, built primarily from another contractile protein, myosin. This part of the sarcomere is denser and forms a darker anisotropic zone (or A-zone).
During contraction, myosin becomes able to interact with actin and begins to pull actin filaments towards the center of the sarcomere (see Fig. 1). As a result of this movement, the length of each sarcomere and the entire muscle as a whole decreases. It is important to note that with this system of motion generation, called the sliding filament system, the length of the filaments (neither actin filaments nor myosin filaments) does not change. Shortening is a consequence only of the movement of the threads relative to each other.
The signal for the start of muscle contraction is an increase in the concentration of Ca 2+ inside the cell. The calcium concentration in the cell is regulated by special calcium pumps built into the outer membrane and the membranes of the sarcoplasmic reticulum, which entwines the myofibrils (see Fig. 1). The above diagram gives a general idea of the mechanism of muscle contraction. To understand the molecular basis of this process, let us turn to an analysis of the properties of the main contractile proteins.
Structure and properties of actin. Actin was discovered in 1948 by Hungarian biochemist Bruno Straub. This protein received its name because of its ability to activate (hence actin) the hydrolysis of ATP catalyzed by myosin. Actin is one of the ubiquitous proteins; it is found in almost all animal and plant cells. This protein is very conserved.
Actin monomers (often called G-actin, that is, globular actin) can interact with each other, forming so-called fibrillar (or F-actin). The polymerization process can be initiated by increasing the concentration of mono- or divalent cations or by adding special proteins. The polymerization process becomes possible because actin monomers can recognize each other and form intermolecular contacts.
Polymerized actin looks like two strings of beads twisted relative to each other, where each bead represents an actin monomer (Fig. 2a). The actin molecule is far from symmetrical, therefore, in order to make this asymmetry visible, part of the actin ball in Fig. 2, b is darkened. The process of actin polymerization is strictly ordered, and actin monomers are packaged into the polymer only in a specific orientation. Therefore, monomers located at one end of the polymer are turned toward the solvent with one, for example, dark end, and monomers located at the other end of the polymer are turned toward the solvent with the other (light) end (Fig. 2, b). The probability of monomer addition at the dark and light ends of the polymer is different. The end of the polymer where the rate of polymerization is greater is called the plus end, and the opposite end of the polymer is called the minus end.
Actin is unique building material, widely used by cells to build various elements of the cytoskeleton and contractile apparatus. The use of actin for the construction needs of the cell is due to the fact that the processes of actin polymerization and depolymerization can be easily regulated using special actin-binding proteins. There are proteins that bind to monomeric actin (for example, profilin, Fig. 2, b). These proteins, being in complex with globular actin, prevent its polymerization. There are special proteins that, like scissors, cut already formed actin filaments into shorter fragments. Some proteins preferentially bind and form a cap (“capped” from English word"cap", cap) at the plus end of the polymer actin. Other proteins cap the minus end of actin. There are proteins that can cross-link already formed actin filaments. In this case, either large-mesh flexible networks or ordered rigid bundles of actin filaments are formed (Fig. 2, b).
All actin filaments in the sarcomere have a constant length and correct orientation, with the plus ends of the filaments located in the Z-disk and the minus ends in the central part of the sarcomere. As a result of this packing, the actin filaments located in the left and right parts of the sarcomere have opposite directions (this is shown in Fig. 1 in the form of oppositely directed checkmarks on the actin filaments in the lower part of Fig. 1).
Structure and properties of myosin. Currently, several (more than ten) have been described various types myosin molecules. Let us consider the structure of the most thoroughly studied skeletal muscle myosin (Fig. 3, a). The skeletal muscle myosin molecule contains six polypeptide chains - two so-called myosin heavy chains and four myosin light chains (LMC). These chains are firmly associated with each other (non-covalent bonds) and form a single ensemble, which is actually a myosin molecule.
Myosin heavy chains have a large molecular weight (200,000-250,000) and a highly asymmetric structure (Fig. 3a). Each heavy chain has a long, coiled tail and a small, compact, pear-shaped head. The helical tails of myosin heavy chains are twisted together like a rope (Fig. 3a). This rope has a fairly high rigidity, and therefore the tail of the myosin molecule forms rod-shaped structures. In several places the rigid structure of the tail is broken. In these places there are so-called hinge regions, which ensure the mobility of individual parts of the myosin molecule. The hinge regions are easily cleaved by proteolytic (hydrolytic) enzymes, which leads to the formation of fragments that retain certain properties of the intact myosin molecule (Fig. 3a).
In the neck region, that is, at the transition of the pear-shaped head of the myosin heavy chain into the spiral tail, short myosin light chains with a molecular weight of 18000-28000 are located (these chains are depicted as arcs in Fig. 3, a). Associated with each myosin heavy chain head are one regulatory (red arc) and one essential (blue arc) myosin light chain. Both myosin light chains in one way or another influence the ability of myosin to interact with actin and are involved in the regulation of muscle contraction.
The rod-shaped tails can stick to each other due to electrostatic interactions (Fig. 3b). In this case, myosin molecules can be located either parallel or antiparallel relative to each other (Fig. 3, b). Parallel myosin molecules are displaced relative to each other by a certain distance. In this case, the heads, together with the myosin light chains associated with them, are located on a cylindrical surface (formed by the tails of myosin molecules) in the form of peculiar protrusions-tiers.
Skeletal muscle myosin tails can pack in either a parallel or antiparallel direction. The combination of parallel and antiparallel packing leads to the formation of so-called bipolar (that is, bipolar) myosin filaments (Fig. 3, b). This filament consists of approximately 300 myosin molecules. Half of the myosin molecules turn their heads in one direction, and the other half - in the other direction. The bipolar myosin filament is located in the central part of the sarcomere (see Fig. 1). The different directions of myosin heads in the left and right parts of the thick filament are indicated by multidirectional checkmarks on the myosin filaments in the lower part of Fig. 1.
The main "motor" part of skeletal muscle myosin is the head of the myosin heavy chain together with the associated myosin light chains. Myosin heads can reach and contact actin filaments. When such contacts close, so-called cross bridges are formed, which actually generate a pulling force and ensure the sliding of actin filaments relative to myosin. Let's try to imagine how such a single cross bridge works.
Modern ideas about the mechanism of functioning of myosin heads. In 1993, isolated and specially modified myosin heads were crystallized. This allowed us to establish the structure of myosin heads and formulate hypotheses about how myosin heads can move actin filaments.
 A - the myosin head is oriented in such a way that the actin-binding center (colored red) is located on the right side. The gap (“open mouth”) separating the two halves (two “jaws”) of the actin-binding center is clearly visible |
It turned out that three main parts can be identified in the myosin head (Fig. 4). N-terminal part of the myosin head with molecular weight about 25,000 (indicated green in Fig. 4, a) forms an ATP-binding center. The central part of the myosin head with a molecular weight of 50,000 (indicated in red in Fig. 4, a) contains an actin binding center. Finally, the C-terminal part with a molecular weight of 20,000 (indicated in purple in Fig. 4, a) forms the framework of the entire head. This part is connected by a flexible hinge to the helical tail of myosin heavy chains (see Fig. 4a). In the C-terminal part of the myosin head there are binding centers for the essential (yellow in Fig. 4, a) and regulatory (light purple in Fig. 4, a) myosin light chains. The general outline of the myosin head resembles a snake with its “mouth” slightly open. The jaws of this “mouth” (colored red in Fig. 4, a) form an actin-binding center. It is assumed that during the hydrolysis of ATP, periodic opening and closing of this “mouth” occurs. Depending on the position of the “jaws,” the myosin head interacts more or less tightly with actin.
Let's consider the cycle of ATP hydrolysis and the movement of the head along actin. In the initial state, the myosin head is not saturated with ATP, the “mouth” is closed, the actin-binding centers (“jaws”) are brought together, and the head interacts firmly with actin. In this case, the spiralized “neck” is oriented at an angle of 45? relative to the actin filament (state 1 in Fig. 4, b). When ATP binds in the active center, the “mouth” opens, the actin-binding sites located on the two “jaws” of the mouth move away from each other, the strength of the connection between myosin and actin weakens, and the head dissociates from the actin filament (state 2 in Fig. 4, b). Hydrolysis of ATP in the active center of the myosin head dissociated from actin leads to the closing of the active center cleft, a change in the orientation of the “jaws” and a reorientation of the spiral neck. After hydrolysis of ATP to ADP and inorganic phosphate, the neck is rotated 45? and occupies a position perpendicular to the long axis of the actin filament (state 3 in Fig. 4b). After all these events, the myosin head is again able to interact with actin. However, if in state 1 the head was in contact with the second actin monomer from the top, now, due to the rotation of the neck, the head engages and interacts with the third actin monomer from the top (state 4 in Fig. 4b). The formation of a complex with actin causes structural changes in the myosin head. These changes allow the release of inorganic phosphate from the active center of myosin, which was formed during the hydrolysis of ATP. At the same time, a reorientation of the neck occurs. It occupies a position at an angle of 45° relative to the actin filament, and during reorientation, a pulling force develops (state 5 in Fig. 4b). The myosin head pushes the actin filament one step forward. After this, another reaction product, ADP, is released from the active site. The cycle closes and the head returns to its original state (state 1 in Fig. 4, b).
Each head generates a small pulling force (a few piconewtons). However, all these small efforts add up, and as a result, the muscle can develop quite large tensions. Obviously, the greater the area of overlap between thin and thick filaments (that is, the more myosin heads that can engage the actin filaments), the greater the force that can be generated by the muscle.
Mechanisms of regulation of muscle contraction. A muscle could not perform its function if it was constantly in a contracted state. For efficient work it is necessary that the muscle have special “switches” that would allow the myosin head to walk along the actin filament only under strictly defined conditions (for example, during chemical or electrical stimulation of the muscle). Stimulation leads to a short-term increase in the concentration of Ca 2+ inside the muscle from 10 -7 to 10 -5 M. Ca 2+ ions are a signal for the start of muscle contraction.
Thus, to regulate contraction, special regulatory systems are needed that could monitor changes in Ca 2+ concentration inside the cell. Regulatory proteins can be located on thin and thick filaments or in the cytoplasm. Depending on where the Ca-binding proteins are located, it is customary to distinguish between the so-called myosin and actin types of regulation of contractile activity.
Myosin type of regulation of contractile activity. The simplest method of myosin regulation is described for some muscles of mollusks. Myosin in mollusks is no different in composition from myosin in vertebrate skeletal muscles. In both cases, myosin contains two heavy chains (with a molecular weight of 200,000-250,000) and four light chains (with a molecular weight of 18,000-28,000) (see Fig. 3). In the absence of Ca 2+ , the light chains are thought to wrap around the hinge region of the myosin heavy chain. In this case, the mobility of the hinge is greatly limited. The myosin head cannot perform oscillatory movements; it is as if frozen in one position relative to the trunk of the thick filament (Fig. 5, a). Obviously, in this state the head cannot carry out oscillatory (“raking”) movements and, as a result, cannot move the actin filament. When Ca 2+ binds, changes in the structure of the light and heavy chains of myosin occur. Mobility in the hinge area increases sharply. Now, after ATP hydrolysis, the myosin head can perform oscillatory movements and push the actin filaments relative to the myosin.
The smooth muscles of vertebrates (such as vascular muscles, the uterus), as well as some forms of non-muscular motility (changes in the shape of platelets), are also characterized by the so-called myosin type of regulation. As in the case of mollusk muscles, the myosin type of regulation of smooth muscles is associated with changes in the structure of myosin light chains. However, in the case of smooth muscles, this mechanism is noticeably more complicated.
It turned out that a special enzyme is associated with myosin filaments of smooth muscles. This enzyme is called myosin light chain kinase (MLCK). Myosin light chain kinase belongs to a group of protein kinases, enzymes capable of transferring the terminal phosphate residue of ATP to the oxy groups of serine or threonine residues of a protein. At rest, at a low concentration of Ca 2+ in the cytoplasm, myosin light chain kinase is inactive. This is due to the fact that the enzyme structure has a special inhibitory (activity-blocking) site. The inhibitory site enters the active center of the enzyme and, preventing it from interacting with the true substrate, completely blocks the activity of the enzyme. Thus, the enzyme seems to put itself to sleep.
 A – hypothetical diagram of the mechanism for regulating muscle contraction in mollusks. One myosin head with light chains and an actin filament in the form of five circles are depicted. In a state of relaxation (a), myosin light chains reduce the mobility of the hinge connecting the head to the trunk of the myosin filament. After Ca 2+ binding (b), the mobility of the hinge increases, the myosin head performs oscillatory movements and pushes actin relative to the myosin. |
In the cytoplasm of smooth muscles there is a special protein calmodulin, which contains four Ca-binding centers in its structure. Ca 2+ binding causes changes in the structure of calmodulin. Calmodulin saturated with Ca 2+ turns out to be able to interact with MLCK (Fig. 5, B). The landing of calmodulin leads to the removal of the inhibitory site from the active center, and myosin light chain kinase seems to wake up. The enzyme begins to recognize its substrate and transfers a phosphate residue from ATP to one (or two) serine residues located near the N-terminus of the myosin regulatory light chain. Phosphorylation of the regulatory myosin light chain leads to significant changes in the structure of both the light chain itself and, apparently, the myosin heavy chain in the region of its contact with the light chain. Only after phosphorylation of the light chain is myosin able to interact with actin and muscle contraction begins (Fig. 5, B).
A decrease in calcium concentration in the cell causes the dissociation of Ca 2+ ions from the cation-binding centers of calmodulin. Calmodulin dissociates from myosin light chain kinase, which immediately loses its activity under the influence of its own inhibitory peptide and again seems to go into hibernation. But while myosin light chains are in a phosphorylated state, myosin continues to carry out cyclic extension of actin filaments. In order to stop the cyclic movements of the heads, it is necessary to remove the phosphate residue from the myosin regulatory light chain. This process is carried out under the action of another enzyme - the so-called myosin light chain phosphatase (MLCM in Fig. 5, B). Phosphatase catalyzes the rapid removal of phosphate residues from the myosin regulatory light chain. Dephosphorylated myosin is not able to carry out cyclic movements of its head and pull up actin filaments. Relaxation occurs (Fig. 5, B).
Thus, both in the muscles of mollusks and in the smooth muscles of vertebrates, the basis of regulation is a change in the structure of myosin light chains.
 Rice. 6. Structural basis of actin-type regulation of muscle contraction |
Actin mechanism for regulating muscle contraction. The actin-related mechanism for regulating contractile activity is characteristic of vertebrate striated skeletal muscle and cardiac muscle. Filaments of fibrillar actin in skeletal and cardiac muscles look like a double string of beads (Fig. 2 and 6, a). The strands of actin beads are twisted relative to each other, so grooves are formed on both sides of the filament. The highly coiled protein tropomyosin is located deep in these grooves. Each tropomyosin molecule consists of two identical (or very similar friends on each other) polypeptide chains that are twisted relative to each other like a girl’s braid. Located within the actin groove, the rod-shaped tropomyosin molecule contacts seven actin monomers. Each tropomyosin molecule interacts not only with actin monomers, but also with the previous and subsequent tropomyosin molecules, as a result of which a continuous strand of tropomyosin molecules is formed within the entire actin groove. Thus, inside the entire actin filament there is a kind of cable formed by tropomyosin molecules.
In addition to tropomyosin, the actin filament also contains the troponin complex. This complex consists of three components, each of which performs characteristic functions. The first component of troponin, troponin C, is capable of binding Ca 2+ (the abbreviation C indicates the ability of this protein to bind Ca 2+). In structure and properties, troponin C is very similar to calmodulin (for more details, see). The second component of troponin, troponin I, was so designated because it can inhibit the hydrolysis of ATP by actomyosin. Finally, the third component of troponin is called troponin T because this protein attaches troponin to tropomyosin. The complete troponin complex has the shape of a comma, the dimensions of which are comparable to the sizes of 2-3 actin monomers (see Fig. 6, c, d). There is one troponin complex per seven actin monomers.
In a state of relaxation, the concentration of Ca 2+ in the cytoplasm is very low. Regulatory centers of troponin C are not saturated with Ca 2+. That is why troponin C weakly interacts with troponin I only at its C-terminus (Fig. 6, c). The inhibitory and C-terminal regions of troponin I interact with actin and, with the help of troponin T, push tropomyosin out of the groove onto the actin surface. As long as tropomyosin is located at the periphery of the groove, the accessibility of actin to the myosin heads is limited. Contact between actin and myosin is possible, but the area of this contact is small, as a result of which the myosin head cannot move along the actin surface and cannot generate a pulling force.
With an increase in the concentration of Ca 2+ in the cytoplasm, the regulatory centers of troponin C become saturated (Fig. 6, d). Troponin C forms a strong complex with troponin I. In this case, the inhibitory and C-terminal parts of troponin I dissociate from actin. Now nothing holds tropomyosin on the actin surface, and it rolls to the bottom of the groove. This movement of tropomyosin increases the accessibility of actin to myosin heads, the area of contact between actin and myosin increases, and myosin heads acquire the ability not only to contact actin, but also to roll along its surface, generating a pulling force.
Thus, Ca 2+ causes a change in the structure of the troponin complex. These changes in troponin structure result in the movement of tropomyosin. Because tropomyosin molecules interact with each other, changes in the position of one tropomyosin will entail the movement of the previous and subsequent tropomyosin molecules. This is why local changes in the structure of troponin and tropomyosin quickly spread along the entire actin filament.
Conclusion. Muscles are the most advanced and specialized device for moving in space. Muscle contraction is accomplished by the sliding of two systems of filaments formed by the main contractile proteins (actin and myosin) relative to each other. Sliding of the filaments becomes possible due to the cyclic closing and opening of contacts between the actin and myosin filaments. These contacts are formed by myosin heads, which can hydrolyze ATP and, due to the released energy, generate a pulling force.
The regulation of muscle contraction is provided by special Ca-binding proteins, which can be located either on the myosin or actin filament. In some types of muscle (for example, vertebrate smooth muscle), the main role belongs to regulatory proteins located on the myosin filament, and in other types of muscle (vertebrate skeletal and cardiac muscle), the main role belongs to regulatory proteins located on the actin filament.
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Reviewer of the article by N. K. Nagradova
Nikolai Borisovich Gusev, Doctor of Biological Sciences, Professor of the Department of Biochemistry, Faculty of Biology, Moscow State University. Area of scientific interests: protein structure, muscle biochemistry. Author of more than 90 scientific papers.
The main muscle proteins are actin and myosin
The mass of muscle fibrils consists of water (75%) and proteins (more than 20%). The main representatives of muscle proteins are actin and myosin, of which myosin accounts for 55%.
This protein (MW 460 kDa) has the form of an asymmetric hexamer. The myosin molecule has an elongated part, consisting of two helices wound one on top of the other. Each helix has a globular head at one end. The hexamer (6 subunits) includes one pair of heavy chains (MW 200 kDa) and two pairs of light chains (MW 15-27 kDa). Heavy chains consist of a linearly elongated, a-helical C-terminal domain (1300 amino acid residues) and a globular N-terminal domain (about 800 amino acid residues). Two a-helical domains belonging to two heavy chains form together a stable superhelical structure with two globular heads (Fig. 17.8).
The complete myosin molecule also contains 4 relatively small polypeptide chains (MW 16-24 kDa), which are associated with globular heads. Unlike actin, skeletal muscle myosin has enzymatic activity and catalyzes the hydrolysis of ATP by binding to F-actin. All light chains bind Ca 2+, are phosphorylated by a special kinase and, in general, take part in the regulation of the activity of myosin ATPase.
Fig.17.8. Schematic representation of the structure of thick filaments. The spatial configuration of myosin is shown.
The myosin molecule contains several functionally important regions. Not far from the middle of the linear supercoiled zone there is a place where the molecule is broken down by trypsin. This enzyme, as it were, cuts the molecule into 2 parts: one contains globular heads and some part of the supercoiled zone; the other consists of the remaining portion of the supercoiled zone at the C-terminus. The part containing the head is called “heavy meromyosin” (MW 350 kDa). The C-terminal fragment is called “light meromyosin” (MW 125 kDa).
The significance of the place of action of trypsin on the myosin molecule is that it surprisingly coincides with the place in the myosin molecule that works as a kind of hinge, turning chemical energy ATP in pure mechanical phenomenon contractions - relaxations. Another important site that performs a similar role is subject to the action of another proteolytic enzyme, papain. Papain cuts the myosin molecule very close to the globular heads. It turns out two fragments and it is the one where the head is located that exhibits ATPase activity.
Thick filaments are formed from myosin. The thick filament consists of approximately 400 myosin molecules, 200 on each side of the M line. These molecules are held together by the C protein (the “clip” protein), the M-line protein, and hydrophobic interactions with each other. At a point localized at the site of trypsin action, heavy meromyosin deviates from the main axis of the thick filament, forming an acute angle. Due to this, the head closely approaches the actin of thin filaments, localized in the space between the thick filaments. The most important molecular event preceding muscle contraction is the regulated binding of myosin heads to thin filament actin. Subsequently, a rapid change in the conformation of myosin occurs around the already mentioned peculiar “hinge” points, and the bound actin moves in the direction of the M-line.
The share of actin in the total mass of muscle proteins is 25%. This is a globular protein monomer with a molecular weight of 43 kDa, called G-actin. In the presence of magnesium ions and the physiological concentration of ions in solution, G-actin polymerizes to form an insoluble filament, which is called F-actin (Fig. 17.9). Two F-actin polymers wrap around each other in a helix. This is how the basic structure of the thin thread is formed. The F-actin fiber has a thickness of 6–7 nm and a repeating structure with a periodicity of 35.5 nm. Neither G- nor F-actin have any catalytic activity.
Rice. 17.9. Structure of F-actin
Each G-actin subunit has an ATP/ADP binding site, which takes part in the polymerization of the thin filament. After polymerization is completed, the thin filament is coated and stabilized by a protein - b-actinin. In addition to the nucleotide binding site, each G-actin molecule has a high-affinity myosin head binding site. Its work in skeletal and cardiac muscles is regulated by additional thin filament proteins. Thus, additional proteins control the contractile cycle.