The role of calcium in the superprecipitation of actomyosin.

In living muscle, calcium appears to be intimately involved in the reactions leading to contraction (l-4). In isolated actomyosin systems (e.g. glycerol-extracted fibers and myofibrils) the addition of calcium will result in contraction under certain conditions: when adenosine triphosphate, magnesium, and one of several relaxing factors are present, e.g. ethylenediaminetetraacetic acid, adenosine triphosphate in excess over magnesium, or the physiological relaxing factor (5-10). All of these compounds strongly bind calcium (11). This fact has recently been demonstrated for the physiological relaxing factor by Ebashil and confirmed and extended by Hasselbach and Makinose (12). When the addition of calcium to an actomyosin system containing magnesium and adenosine triphosphate in the presence of one of the relaxing factors results in contraction, at least two different interpretations of the mode of action of calcium are possible. Calcium might combine with actomyosin, a complex between actomyosin and calcium being necessary for contraction. The inhibition of contraction before the addition of calcium would then have been due to the removal of calcium from actomyosin by the relaxing factor (13). This possibility has been considered also by Ebashi (14) and Baird and Perry (15). Or it might be considered that calcium interferes with a reaction between one of the relaxing factors and actomyosin (16-18). On this assumption, contraction is inhibited by relaxing factors because they combine with actomyosin. For further clarification of this question we studied the effect of the removal of calcium on the superprecipitation and adenosine triphosphatase activity of actomyosin. We consider superprecipitation and high adenosine triphosphatase activity to be comparable to contraction, and inhibition of superprecipitation together with a low adenosine triphosphatase activity to be comparable to relaxation. We used for our investigation “synthetic” actomyosin prepared from purified actin and myosin in order to be reasonably sure that the system was free from endoplasmic reticulum, the physiological relaxing factor (19). It has been found by inspection with the electron microscope that glycerol-extracted fiber bundles, well washed myofibrils, and “natural” actomyosin, but not “synthetic” actomyosin, still contain remnants of endoplasmic reticulum.2 Such a contamination should be avoided to exclude the possibility that the effects of calcium and calciumchelating agents on actomyosin are indirect, i.e. mediated through their influence on the physiological relaxing factor.