Myosin-actin binding: One of the biologically important properties of myosin is its ability to combine with actin. The complex formed is called actomyosin. The actin binding by myosin is highly specific; no other protein can substitute actin. Physiologically, when actin and myosin combine the muscle produces force.

There are several methods to measure the stoichiometry of actin to myosin combination. In solution, analytical ultracentrifugation is the most accurate method: When molecules in solution are subjected to high centrifugal field, they sediment according to their molecular weight. Runs in the analytical ultracentrifuge were performed in our laboratory with solutions containing the same amount of myosin (M) and different amount of fibrous actin. As Fig. M4 shows, without actin only 1 peak, the myosin peak, is seen in the analytical ultracentrifuge. As actin is added to myosin in increasing amount, part of the myosin combines with actin, forming the high molecular weight actomyosin (AM) and 2 peaks are seen. When all the myosin combined with actin, again only 1 peak, the actomyosin peak, is seen.

Viscometry is another method to measure myosin-actin binding in solution. The viscosity of actomyosin is much higher than the sum of its individual components actin and myosin. Thus, when constant amount of myosin is titrated with increasing amount of fibrous actin the stoichiometry of the actin binding is reached at the maximal viscosity.

In suspension, at physiological ionic strength, the myosin-actin binding is measured under conditions when both myosin and actomyosin are insoluble, that is they sediment at low centrifugal fields. Thus, adding increasing amount of fibrous actin to constant amount of myosin in suspension will result in actomyosin formation. After myosin becomes saturated with actin, the uncombined actin remains in the supernatant of the centrifugate.

Results from either solution or suspension studies show that 1 myosin molecule binds 2 molecules of globular actin units in the fibrous actin polymer, or since each myosin molecule has two heads (where the actin-binding site resides) each myosin head combines with one molecule of globular actin.

ATPase activity of myosin: A Russian husband wife team, Engelhardt and Lyubimova, made the important discovery in 1939 that myosin is an enzyme that hydrolyzes ATP. It was already known that ATP is the universal energy donor in living cells, thus Engelhardt and Lyubimova created the term mechanochemistry i.e. the contractile protein myosin that carries out the work also liberates the energy necessary for the work. This idea is widely accepted today.

The ATPase activity of rabbit skeletal myosin at 37oC in an ionic medium resembling the intracellular fluid of resting muscle (100 mM K+, 2 mM free Mg2+, and 0.1 pM Ca2+) is low, about 0.2 mole of inorganic phosphate (Pi) is liberated per mole myosin head per second. Actin is the physiological activator of myosin ATPase, the substrate is MgATP2-. During muscle contraction the Ca2+ concentration in the intracellular fluid is increased to about 10 pM and the ATPase activity of myosin activated by actin is increased 50-100 times to about 10-20 mole of Pi per mole myosin head per second.

ATP is hydrolyzed by myosin also in the presence of 10 mM Ca2+; this is not physiological. Furthermore, myosin hydrolyzes ATP in the absence of bivalent metals, i.e. in the presence of EDTA, a strong complexing agent for Mg2+ and Ca2+, but K+ has to be present. Na+ cannot substitute K+ . This (K+ + EDTA) ATPase is not physiological either, but it is useful for detection of myosin in non-muscle systems.

The dependence of various ATPase activities of skeletal muscle myosin on the KCl concentration and pH is sketched in Fig. M5.

Fig. M4. Myosin-actin binding as followed in the analytical ultracentrifuge.

Separate actin-binding and ATPase sites of myosin: Titration of the cysteine residues of myosin revealed that the actin-binding ability of myosin can be separated from its ATPase activity (Fig. M5a). These results indicate that the sites of myosin that interact with actin and ATP are different.

Fig. M5a. Separation of the actin-binding ability of myosin from its ATPase activity. Abscissa shows the remaining cysteine residues (SH groups) of myosin after titration with iodoacetamide. Ordinate shows the percentage of actin-binding ability and ATPase activity of the treated myosin relative to those of the control. Symbols: x-x-x, actin-binding ability; open and filled squares, triangles and circles refer to the Ca2+- and actin-activated ATPase activities at various pH values. (From Barany and Barany, 1959).

Intermediates of the ATP hydrolysis: It was shown first by Taylor (Lymn and Taylor, 1970; Taylor et al., 1970) that the ATP hydrolysis catalyzed by myosin involves several intermediates:

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