Interaction of myosin filaments and minifilaments with actin: a comparative study.

Various aspects of actin-myosin interaction were investigated using myosin in the form of filaments and minifilaments obtained by dialysis against citrate-Tris buffer or by adding this buffer to performed myosin filaments. Considerable similarities in the behaviour of the two systems were found. (1) Although the minifilaments are soluble structures, they form insoluble complexes with actin, which superprecipitate upon addition of MgATP. Observations in the electron microscope and from centrifugation experiments have shown that the two actomyosin systems undergo essentially similar structural changes during superprecipitation. (2) At low substrate concentrations the rate of ATP hydrolysis in both systems declines with time, which is typical of insoluble superprecipitating actomyosin. (3) In contrast to soluble myosin subfragments, both filamentous and minifilamentous myosin give biphasic actin-activation curves. (4) The Mg2+-ATPase activities of myosin minifilaments and standard myosin preparations at low KCl extrapolate to similar Vmax at infinite actin concentration. Since our values of Vmax for myosin filaments and minifilaments are in the range of those reported for myosin subfragments, the results of this investigation confirm the view that the catalytic properties of myosin subfragments and intact myosin are equivalent. Moreover, the data show that the extent of myosin aggregation in the initial preparations has no appreciable effect on the characteristic features of the interaction between intact myosin and actin at pH 8.

Unusual features of the Ca2+-ATPase activity of myosin from fast skeletal muscle of the frog: effect of actin and SH1 thiol group modification.

The K+-ATPase and actin-activated Mg2+-ATPase activity of myosin from fast skeletal muscle of the frog, Rana esculenta or Rana temporaria, are comparable to the respective activities of rabbit fast skeletal muscle. On the other hand, the Ca2+-ATPase activity of the same preparations of frog myosin is 6-7-fold lower than that of myosin from rabbit muscle. Various control experiments indicate that the small extent of Ca2+ stimulation is an intrinsic property of frog muscle myosin. Unlike myosin from rabbit muscle, the Ca2+-ATPase activity of frog myosin is strongly activated by actin; at high actin concentrations it approaches the level of the Ca2+-ATPase activity of rabbit myosin. The levels of Ca2+-ATPase activity of frog and rabbit myosins also become comparable upon modification of myosin SH1 thiol groups; this means that the modification of the SH1 groups results in a much higher activation of the Ca2+-ATPase of frog myosin than that of rabbit myosin. The results suggest a difference in the active site conformation in frog and rabbit muscle myosins. The effects of actin and SH1 group modification are discussed in terms of allosteric changes which diminish the difference in the active site conformation of the two myosins. We have also observed a difference in the reactivity of thiol groups which are not essential for the enzymatic activity in frog and rabbit myosin, indicating structural differences in regions other than the active site.

Changes in the ultrastructure of actomyosin gel during hydrolysis of ATP under various ionic conditions.

The interaction of myosin and actin in the presence of MgATP under various ionic conditions was investigated by a simultaneous determination of changes in turbidity, liberation of inorganic phosphate, distribution of the two proteins between the supernatant and precipitate obtained after a short-time centrifugation, and by electron microscopy. The results confirm the view that the extent of association between myosin and actin filaments is low not only in the clear phase but also under the conditions when the addition of MgATP results in superprecipitation without a detectable clear phase. Extensive formation of aggregates of parallelly aligned myosin and actin filaments coincides with the depletion of ATP, indicating that these aggregates represent rigor complexes. Relation between ATP-induced turbidity changes and structural changes in the actomyosin system under various ionic conditions is discussed.