Protein Chemistry - Part 2 - Selected Topics in Special Protein Chemistry - Ashmarin, I. P. 1968

Contractile proteins of muscle tissue
Myosin

Physicochemical properties and composition of myosin. Myosin is a protein with a relatively high molecular weight, ranging from 450,000 to 500,000. It should be noted that myosin has a complete amino acid profile, containing all known Amino Acids. The proportion of dicarboxylic (18%) and basic (16%) amino acids is particularly high. An N-terminal amino acid has not yet been identified, whereas the C-terminal is represented by isoleucine.

Its molecule possesses a high degree of Asymmetry: it is about 1400 A in length and 20–28 A in diameter. Electron micrographs show it as a rod-like Structure with two globular formations at one of its ends (Fig. 4).

The Characteristic Properties of myosin solutions—Streaming Birefringence and high viscosity—are consequences of its molecular asymmetry and high molecular weight. The viscosity of pure myosin solutions is non-anomalous, meaning it is practically independent of pressure (within the range of 5–100 cm of Water Column) and is related to its concentration by a nearly linear dependence. Thixotropy is also absent. This indicates the lack of significant additional intermolecular interaction forces in solution that are so characteristic of other Muscle Proteins. The solubility of myosin in water is characterized by a complex dependence on the Ionic strength of the medium. Salt-free myosin gel is soluble in water. However, the presence of even 0.001 M potassium chloride sharply reduces myosin solubility, and at 0.02–0.05 M it precipitates completely. Its solubility remains very low up to a potassium chloride concentration of 0.15 M. It is worth noting that the concentration of potassium chloride within the myofibril is precisely 0.15 M, which consequently corresponds to the gel-like state of the bulk of the myosin (not to mention the precipitating effect of Actin, magnesium ions, and Calcium Ions present in the myofibril). With a further increase in potassium chloride concentration, myosin solubility increases, reaching a maximum at 0.4–0.6 M. Myosin is salted out by ammonium sulfate at 48–49% solution saturation. Its solubility in the absence of salts and the conditions of precipitation with ammonium sulfate allow myosin to be classified among pseudoglobulin fractions. However, the insolubility of myosin at potassium chloride concentrations close to or lower than physiological levels is its distinct feature. Myosin gels are characterized by an extremely high water content (95–98%), making it highly hydrophilic. The phenomenon of myosin precipitation at low potassium chloride concentrations is attributed to The formation of electroneutral potassium myosinate; in the absence of cations, its isoelectric point lies in a mildly acidic environment—at pH 5.4—indicating the presence of only a relatively small number of excess electronegative groups. Divalent magnesium and calcium ions also precipitate myosin even at a concentration of 0.001 M.

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Fig. 4. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF myosin molecules (Lowey et al., 1969).

Adenosine triphosphatase activity of myosin. As already noted, establishing the adenosine triphosphatase activity of myosin was of paramount importance for uncovering the chemical mechanisms of Muscle contraction. Years of research in this field have fully confirmed that the enzymatic activity is inseparable from the myosin molecule. Although its specific activity is low (Hydrolysis of about 50 moles of ATP per 1 mole of myosin per 1 sec), its significance is great because it is within the myosin molecule that the crucial stages of converting the chemical energy released during ATP hydrolysis into mechanical energy take place, and because The amount of myosin in Muscle tissue is relatively very large. The ATPase activity of myosin is highly specific in that only the third terminal phosphate group of ATP is cleaved. Myosin exhibits neither ADPase nor AMPase activity. Myosin lacks Specificity regarding the nucleoside radical linked to the triphosphate group; it hydrolyzes nucleoside triphosphates such as GTP, UTP, CTP, and ITP even more vigorously, and finally, it hydrolyzes simple inorganic triphosphate, albeit significantly slower than ATP. The fact that ATP, rather than other nucleoside triphosphates, serves as the direct energy source for contraction is presumably determined by its general role as the universal energy donor in biochemical reactions.

Metal Ions play an important role in the manifestation of adenosine triphosphatase activity. In the absence of cations, myosin is inactive. Potassium ions activate myosin, but the activating effect of calcium ions is particularly characteristic. In the presence of potassium chloride at concentrations of 0.05–0.15 M, i.e., close to or lower than physiological levels, ATPase activity increases significantly even with just 0.001 M calcium chloride present. Conversely, magnesium ions, also at a concentration of 0.001 M, sharply inhibit myosin activity (both independently and in the presence of potassium and calcium ions). The dependence of myosin ATPase activity on pH is characterized by two maxima: a major one at pH 9.0 and a less pronounced one at pH 6.3–6.5. At physiological pH values of 7.0–7.4, the activity is also quite substantial—about 40% of the maximum (Fig. 5).

The structure of the region of myosin responsible for ATPase activity is gradually being elucidated. Certain types of SH groups of myosin are of particular importance. Not all SH groups are associated with enzymatic activity, which is manifested in the dual Nature of the action of various specific SH Reagents. For instance, an SH-blocking agent such as p-chloromercuribenzoate even activates myosin at low concentrations, whereas it inhibits it at high concentrations.

Recent studies suggest that myosin contains Two Types of SH groups associated with ATPase activity. Blocking one of them completely inactivates myosin, while blocking the other disrupts the ability of myosin ATPase to be activated by calcium ions. The Amino Acid Sequence near these SH groups has been elucidated:

Fig. 5. Dependence of myosin ATPase activity on medium pH (Engelhardt, 1946).

1 — calcium salt of ATP; 2 — the same in the presence of 0.01 M magnesium chloride.

Thus, we are approaching the elucidation of the Introduction/19.html">Primary Structure of myosin in the region of its active center. Each myosin molecule contains two such centers. Finally, THE POSITION OF the active center has been established at one of the ends of the rod-shaped myosin molecule, within the aforementioned globular regions (see Fig. 4, as well as Fig. 6).

Fig. 6. Schematic diagram of the myosin molecule (Lowey et al., 1969).

LMM - light meromyosin, HMM - heavy meromyosin, HMM S-1 and HMM S-2 - First and Second subfragments of heavy meromyosin, respectively.

Special mention should be made of the instability of pure myosin to various Denaturing Agents. In particular, brief (20-minute) exposure of myosin at 40° C sharply reduces its ATPase activity and significantly impairs its ability to bind actin. Rapid inactivation is also observed in the presence of trace concentrations of heavy metal ions. Conversely, the stability of myosin is considerably enhanced upon complexation with ATP and actin.

Interaction of myosin with actin. The next crucial property of myosin is its ability to form stable complexes with another contractile protein, actin. However, the formation and properties of these complexes are best discussed after the reader has familiarized themselves with the characteristics of not only myosin, but actin as well. Furthermore, it is advisable to dedicate a separate paragraph (§ 5) to the description of Actomyosin, as its properties are directly related to the Mechanism of muscle contraction.

Here we note only that an important role in the interaction with actin is played by SH-groups located in the globular region of the myosin molecule. It is hypothesized that these SH-groups are identical to those responsible for the adenosine triphosphatase activity of myosin.

Details of the myosin molecule structure. Over the past decade, important new data have been obtained regarding the structural details of the myosin molecule. It has been found that upon brief Treatment (7–12 min) with A number of proteinases—Trypsin, Chymotrypsin, subtilisin, Papain—which cleave about 60 peptide bonds, the myosin molecule breaks down into two fragments, the so-called meromyosins. This indicates the presence of highly sensitive sites that readily react with proteinases.

Meromyosins differ in molecular weight and are designated as heavy meromyosin (HMM) and light meromyosin (LMM). Heavy meromyosin has a molecular weight close to 300,000. It retains ATPase activity and The ability to bind actin—properties inherent to the native myosin. At the same time, it exhibits a reduced capacity for forming ordered structures in gels, a characteristic feature of intact myosin. This is manifested in the inability to form crystals (or pseudocrystals) and in the absence of the characteristic precipitation of myosin in 0.02–0.05 M potassium chloride solutions. Further Digestion of heavy meromyosin with proteinases (papain) allows it to be separated into three subfragments, two of which are identical and commonly designated as HMM S-1, while one differs significantly in properties and is designated as HMM S-2. The first two subfragments constitute precisely the globular domains of the myosin molecule observed in electron micrographs (see Fig. 4). They are the bearers of both ATPase activity and the actin-binding capacity. The Molecular Weight of each is 120,000, and the globule diameter is close to 70 Å. Characteristically, the polypeptide chain comprising the globule exists in an a-helical conformation for only a minor fraction of its length.

The HMM S-2 subfragment is a rod-like particle consisting of two polypeptide chains predominantly in an a-helical conformation. Its molecular weight is 60,000, and its length is about 500 Å. It is believed that this fragment forms the most flexible part of the myosin molecule, linking the linear structures that slide past each other During muscle contraction—namely, L-meromyosin and the complex of actin with the HMM S-1 subfragment.

Light meromyosin (LMM) is a rod-like particle with a molecular weight of 150,000 and a length of about 800 Å. Similar to HMM S-2, it consists of two polypeptide chains in the a-conformation. L-meromyosin lacks enzymatic activity and affinity for actin; it confers upon myosin the ability to form ordered structures within gels and myofibrils. Unlike heavy meromyosin, it precipitates in 0.02–0.05 M potassium chloride and can form needle-like crystals.

The sum of the molecular weights of the meromyosins (450,000) is close to the molecular weight of the original myosin. The total length of the meromyosin particles (the first being 600 Å, the second 800 Å) also corresponds to the length of the intact myosin molecule (1400 Å). However, a comprehensive Analysis of the products of mild myosin proteolysis suggests that, In addition to the major components—H- and L-meromyosins—myosin contains yet another component with a low molecular weight of about 20,000. This component (commonly designated as LMP) is bound to the rest of the myosin molecule by non-covalent interactions and can be dissociated by treatment with 1–3 M urea or guanidine, as well as at high pH (around 11). Its complete removal leads to the loss of both ATPase activity and actin-binding capacity in myosin. Meanwhile, the LMP component on its own does not possess these properties.

Let us now examine the overall STRUCTURE OF THE myosin molecule. It is presented in Fig. 6, which shows the sequential arrangement of the fragments and subfragments described above. The diagram also clearly shows that the myosin molecule as a whole consists of two polypeptide chains that are closely associated with each other in the L-meromyosin and the subsequent H-meromyosin subfragment. At the end of the molecule, the strands diverge to form globular domains. It is noteworthy that myosin is dual in nature, combining both globular and fibrillar components in its structure. Obviously, this reflects the functional features of myosin, which not only hydrolyzes ATP but also acts as a transducer, converting the chemical energy released in the process into mechanical work. Within the myofibril, the myosin molecules are oriented with their L-meromyosin components facing the M-line, and their H-meromyosin components facing the edges of the anisotropic disc, where interaction with actin and ATP takes place.