Myosin and

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

Proteins can be broadly classified into fibrous and globular. Many fibrous proteins serve a stmctural role (11). CC-Keratin has been described. Fibroin, the primary protein in silk, has -sheets packed one on top of another. CoUagen, found in connective tissue, has a triple-hehcal stmcture. Other fibrous proteins have a motile function. Skeletal muscle fibers are made up of thick filaments consisting of the protein myosin, and thin filaments consisting of actin, troponin, and tropomyosin. Muscle contraction is achieved when these filaments sHde past each other. Microtubules and flagellin are proteins responsible for the motion of ciUa and bacterial dageUa. 

Alpha helices are sufficiently versatile to produce many very different classes of structures. In membrane-bound proteins, the regions inside the membranes are frequently a helices whose surfaces are covered by hydrophobic side chains suitable for the hydrophobic environment inside the membranes. Membrane-bound proteins are described in Chapter 12. Alpha helices are also frequently used to produce structural and motile proteins with various different properties and functions. These can be typical fibrous proteins such as keratin, which is present in skin, hair, and feathers, or parts of the cellular machinery such as fibrinogen or the muscle proteins myosin and dystrophin. These a-helical proteins will be discussed in Chapter 14. 

Muscle fibers contain myosin and actin which slide against each other during muscle contraction... 

In addition to the major proteins of striated muscle (myosin, actin, tropomyosin, and the troponins), numerous other proteins play important roles in the maintenance of muscle structure and the regulation of muscle contraction. Myosin and actin together account for 65% of the total muscle protein, and tropomyosin and the troponins each contribute an additional 5% (Table 17.1). The other regulatory and structural proteins thus comprise approximately 25% of the myofibrillar protein. The regulatory proteins can be classified as either myosin-associated proteins or actin-associated proteins. 

The molecular events of contraction are powered by the ATPase activity of myosin. Much of our present understanding of this reaction and its dependence on actin can be traced to several key discoveries by Albert Szent-Gyorgyi at the University of Szeged in Hungary in the early 1940s. Szent-Gyorgyi showed that solution viscosity is dramatically increased when solutions of myosin and actin are mixed. Increased viscosity is a manifestation of the formation of an actomyosin complex. 

Figure 7. Electron micrograph of cross section of Drosophi/a leg hexagonal lattice arrangement of thick (myosin) and tl " filaments appear hollow in this inse —...

In this chapter we will discuss the various forms of myosin and the roles they play in living systems. We will compare and contrast the function and regulation of myosin activity in different cellular environments. Finally, we will examine the clinical aspects of myosin strucmre and function. 

In a previous section we mentioned the significance of myosin filament structure. In nematodes two forms of myosin-II, myosin A and B, are required for proper filament stmcture (Epstein, 1988). The two forms of myosin are expressed at the proper time to allow for correct filament assembly. An accessory protein called paramyosin is also required for correct filament assembly. In vertebrate cardiac muscle, there are also two isoforms of myosin-II a-myosin and p-myosin. The proper ratio of these two proteins is of utmost importance for proper muscle activity. The incorrect synthesis of a- and P-myosins results in a severe cardiac disorder known as hypertrophic cardiomyopathy. Genetic transmission of the disease occurs in about 55% of families. The inherited condition is called familial hypertrophic cardiomyopathy (FHC), and this condition is a leading cause of sudden death in young athletes. 

Adelman, M.R., Taylor, E.W. (1969). Further characterization of slime mold myosin and slime mold actin. Biochemistry, 8, 4976-4988. 

Inside the typical smooth muscle cell, the cytoplasmic filaments course around the nuclei filling most of the cytoplasm between the nuclei and the plasma membrane. There are two filamentous systems in the smooth muscle cell which run lengthwise through the cell. The first is the more intensively studied actin-myosin sliding filament system. This is the system to which a consensus of investigators attribute most of the active mechanical properties of smooth muscle. It will be discussed in detail below. The second system is the intermediate filament system which to an unknown degree runs in parallel to the actin-myosin system and whose functional role has not yet been completely agreed upon. The intermediate filaments are so named because their diameters are intermediate between those of myosin and actin. These very stable filaments are functionally associated with various protein cytoarchitectural structures, microtubular systems, and desmosomes. Various proteins may participate in the formation of intermediate filaments, e.g., vimentin. 

The structure of the contractile apparatus of smooth muscle at the next higher level is also characteristically different from other muscles. The concentrations of actin and myosin in smooth muscle are about three times higher for actin and four times lower for myosin than in skeletal muscle. Correspondingly, in smooth muscle the ratio of the numbers of moles of actin to moles of myosin, and the ratio of the number of actin filaments to those of myosin filaments, are about 12 times larger than for other muscles. Thus, the arrangements of the two sets of filaments are bound to be quite different just on the basis of numbers of actin and myosin... 

In order to exert a force from one end of a muscle to the other, a structure must be continuous from one end to the other. In smooth muscle tissue, this structure is a system of alternating myosin and actin filaments within a cell, firm attachments first to the cell membrane, and beyond that extracellular attachments to the... 

Of the several kinase activities which are important in smooth muscle, myosin light chain kinase, MLCK, is the one responsible for activation of the actin-myosin system to in vivo levels. MLCK is present in the other nonmuscle cell types which have the actin-myosin contractile system and all of these are probably activated in a manner similar to smooth muscle rather than by way of the Ca -troponin mechanism of striated muscle. MLCK from smooth muscle is about 130 kDa and is rather variable in shape. It is present in smooth muscle in 1-4 pM concentrations and binds with an equally high affinity to both myosin and actin. Thus, most MLCK molecules are bound to actin. Myosin light chain serine-19 is the primary target of smooth muscle myosin light chain kinase. 

Although in in vivo circumstances an intracellular free calcium increase apparently acts as the primary modulator of contraction, it can be bypassed in highly permeabilized smooth muscle preparations where the active subunit of MLCK can be introduced to phosphorylate myosin and induce contraction. The MLCK catalyzed phosphorylation of serine-19 is seen as the necessary event in the activation of smooth muscle myosin to form crossbridges. Thus, the rising phase of force during an isometric smooth muscle contraction follows an increase in the degree of phosphorylation of myosin, and that in turn follows the transient rise of (a) cytosolic free Ca, (b) Ca-calmodulin complexes, and (c) the active form of MLCK. The regulation of the intracellular calcium is discussed below. The dynam-... 

As ATP binding to myosin, and ATP hydrolysis, are both faster than the overall observed ATPase rate, the slow step that follows the rapid phosphate burst and that must limit the overall observed ATPase rate must be the release of phosphate, or the release of ADP. The rate at which ADP is released was measured by a displacement technique (Trentham et al., 1972), in which the rate at which ADP bound to S-1 is displaced by ATP was measured. This experiment showed that the rate of ADP release (2 s ) is greater than the overall rate of hydrolysis (0.03 s" ). Thus the release of Pj, and not that of ADP, is rate limiting. 

Lowey, S., Goldstein, L., Cohen, C., Luck, S.M. (1966). Proteolytic degradation of myosin and the meromyosins by a water-insoluble polyanionic derivative of trypsin. J. Mol. Biol. 23,287-304. 

Voluntary muscle contraction is initiated in the brain-eliciting action potentials which are transmitted via motor nerves to the neuromuscular junction where acetylcholine is released causing a depolarization of the muscle cell membrane. An action potential is formed which is spread over the surface membrane and into the transverse (T) tubular system. The action potential in the T-tubular system triggers Ca " release from the sarcoplasmic reticulum (SR) into the myoplasm where Ca " binds to troponin C and activates actin. This results in crossbridge formation between actin and myosin and muscle contraction. 

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