Adenosine triphosphatase activity and superprecipitation of canine cardiac myosin B.

Abstract

The changes in the filament structures of dog cardiac myosin B induced by ATP were studied by electron microscopy. At higher ionic strengths a polarized “arrowhead” structure was seen, which disappeared on addition of ATP, while the ordinary actin structure appeared. At lower ionic strengths, myosin aggregates were seen besides actin filaments in the presence of a high concentration of ATP, where myosin B showed a clearing response, while in the presence of a low concentration of ATP, where myosin B showed superprecipitation, many large aggregates were formed. The kinetics of the ATPase and superprecipitation of dog cardiac myosin B were investigated under different conditions. The extent and rate of superprecipitation of cardiac myosin B induced by ATP remained at constant levels above a critical myosin B concentration. Below this the extent was proportional to the concentration of myosin B, but the specific rate decreased with decrease in the myosin B concentration and superprecipitation did not occur below a critical protein concentration. Both the rate of superprecipitation and the ATPase activity of cardiac myosin B decreased with increasing KC1 concentration up to a critical concentration, above which superprecipitation did not occur and the ATPase activity decreased to a level almost equal to that of myosin alone. The minimum concentration of ATP required to maintain the maximum extent of superprecipitation decreased on addition of pyruvate kinase and hosphoenolpyruvate, and the minimum amount of ATP required in the presence of a sufficient amount of the kinase was about one mole, or less than one mole, per mole of cardiac myosin. Over a wide range of ATP concentration, the ATPase activity and superprecipitation of cardiac myosin B were sensitive to Ca++ and EGTA, and the ATPase activity and the extent and rate of superprecipitation increased as the ATP concentration increased until a critical concentration was reached. Above this the clearing response and the substrate inhibition of ATPase took place. The substrate inhibition of cardiac myosin B ATPase was less marked than that of the skeletal myosin B ATPase, particularly in the presence of Ca++, and the clearing response was often accompanied by a high actomyosin type ATPase activity. Even in the presence of a large excess of ATP, where myosin B showed a typical clearing response, the ATPase activity of cardiac myosin B was several times greater than that of cardiac myosin alone in the presence of either Ca++ or EGTA. With aging of the preparation of cardiac myosin B, the superprecipitation and ATPase activity were very rapidly desensitized to Ca++ and EGTA, and the critical concentration of ATP required to induce the substrate inhibition of the ATPase of aged cardiac myosin B was markedly higher than that for a fresh preparation. In the presence of the minor component of metin from skeletal muscle, the ATPase activity and superprecipitation of an aged preparation of cardiac myosin B were sensitized to Ca++ and EGTA. The kinetics of the ATPase of cardiac myosin, viz, the constituent of myosin B, was also studied. The amount of the initial extra-liberation of Pi from the cardiac myosin—ATP system was about one nole per mole of myosin. At the steady state the cardiac myosin ATPasc reaction followed the Michaelis-Menten kinetics, and the Michaelis constant and the maximum rate for cardiac myosin ATPase in 0.5 M KC1—2 mM MgCl2—0.8 mM phosphoenolpyruvate—0.15 mg. pyruvate kinase per ml. at pH 7.0 and 25°C were found to be 3.5 μM and 2.6 μmoles g.−1 min−1, respectively.