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.

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

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 µM 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 µM 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. M5. KCl and pH dependency of myosin ATPase activity.

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. In the three-dimensional structure of the myosin head (Fig. M9) the localization of the separate sites can be seen.

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 Bárány and Bárány, 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:

Subsequent work by Taylor and others revealed that the above reaction is much more complex than shown in the equation. Part b of Fig. M6 shows a recent version of the ATPase cycle and Part a of Fig. M6 shows the coupling of the ATPase cycle to the crossbridge cycle.

Fig. M6. Crossbridge cycle (Part a) correlated with the ATPase cycle (Part b). (From Perry, 1996).

ATPase activity of myosin and speed of muscle shortening: The ATPase activity of myosin was determined in 25 different muscles with a 250-fold variation in the speed of shortening. A correlation was found between the ATPase activity of myosin and the speed of shortening (Fig. M7). This suggests that the myosin ATPase determines the speed of muscle shortening (Bárány, 1967).

Fig. M7. Relationship between myosin ATPase activity and speed of muscle shortening.

Cross-innervation studies provided further evidence for a physiological role of the myosin ATPase - muscle shortening relationship (Bárány and Close, 1971). In rat, the fast extensor digitorum longus muscle was cross innervated with the nerve of the slow soleus muscle and vice versa. This transformed the fast extensor to a slow muscle and the slow soleus to a fast muscle. The changes in the myosin ATPase activity closely followed the changes in the speed of shortening of the cross-innervated muscles (Fig. M8). The high actin-activated ATPase activity of myosin from the extensor muscle was reduced to the low ATPase activity of myosin from the soleus. At the same time the myosin ATPase activity of the cross-innervated soleus was elevated to the level of the normal extensor. A new type of myosin is synthesized in the cross-innervated muscle, which carries the genetic information necessary to determine the speed of muscle contraction.

Fig. M8. The relationship between speed of muscle shortening and actin-activated ATPase activity of myosin in cross-innervated muscles. The symbols represent normal or cross-innervated extensor digitorum longus and soleus muscles.

Myosin heavy chains: The myosin heavy chain (MHC) of different skeletal muscles exhibits diversity. MHC from slow muscle has a slightly lower molecular weight than MHC from the fast muscle, as assessed by SDS-PAGE. The MHC isoform expressed in a single muscle fiber is correlated with the contraction speed of the fiber (Reiser et al., 1985).

During development of the muscle, its contractile properties and myosin isozyme composition are changing. As the muscle speed is increasing more of the fast type MHC is incorporated into the fibers. Furthermore, changes in MHC composition were demonstrated upon increased use of muscle, resulting in hypertrophy, or upon denervation causing atrophy.

Three-dimensional structure of subfragment 1: Rayment and collaborators crystallized myosin subfragment 1 and determined its three-dimensional structure by x-ray diffraction at 2.8-Å resolution (Rayment et al., 1993). A space-filling model was constructed that is illustrated in Fig. M9. In the picture, the left corresponds to the head-tail junction and the right to the top of the head where actin binds. The 25-kDa and 50-kDa fragments of S1 form the ATPase site and the 50-kDa and 20-kDa fragments form the actin-binding site. The ATP-binding site has the sequence GLY-GLU-SER-GLY-ALA-GLY-LYS-THR, which is similar to the sequences found in the active sites of other ATPases. The ATP-binding site was identified as a pocket. There is a cleft in the upper part of the head that extends from under the ATP-binding site to the actin interface. Both portions, above and below the cleft are involved in the actin binding. When ATP binds to S1, the pocket most likely closes and the cleft widens disturbing the S1-actin binding, that is ATP dissociates S1 from actin. When ATP is hydrolyzed by S1 to ADP and Pi, actin recombines with S1. The accompanying structural changes are the narrowing of the cleft and opening of the ATP-binding pocket. These subtle changes are called conformational changes that play a key role in the mechanism of muscle contraction.

The regulatory and essential light chains wrap around the 20-kDa fragment of S1, which forms an 85Å-long a helix that spans much of the S1. Rayment suggested that the conformational changes in the nucleotide-binding pocket could be transmitted to the 85Å-long a helix. This structure may play the role of a lever arm, which magnifies small conformational changes to larger movements. The lever arm hypothesis is presented in the Actin-Myosin Interaction chapter.

Fig. M9. Space-filling model of S1 (From Rayment et al., 1993).


Bárány, M. (1967). ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50, 197-218.

Bárány, M. and Bárány, K. (1959). Studies on "active centers" of L-myosin. Biochim. Biophys. Acta, 35, 293-309.

Bárány, M. and Close, R. I. (1971). The transformation of myosin in cross-innervated rat muscles. J. Physiol. 213, 455-474.

Engelhardt, V.A. and Lyubimova, M.N. (1939). Myosin and adenosinetriphosphatase. Nature, 144, 668.

Lymn, R.W. and Taylor, E.W. (1970). Transient state phosphate production in the hydrolysis of nucleoside triphosphates by myosin. Biochemistry, 9, 2975-2983.

Perry, S.V. (1996). Molecular mechanisms in striated muscle. Cambridge University Press.

Rayment, I., Rypniewski, W.R., Schmidt-Bäse, K., Smith, R., Tomchick, D.R., Benning, M.M., Winkelman, D.A., Wesenberg, G., and Holden, H.M. (1993). Three-dimensional structure of myosin subfragment-1: a molecular motor. Science, 261, 50-58.

Reiser, P.J., Moss, R.L., Giulian, G.G., and Greaser, M.L. (1985). Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. J. Biol. Chem. 260, 9077- 9080.

Taylor, E.W., Lymn, R.W., and Moll, G. (1970). Myosin-product complex and its effect on the steady-state rate of nucleoside triphosphate hydrolysis. Biochemistry, 9, 2984-2991.

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