Studies on Myofibrillar Adenosine Triphosphatase with Calcium-free Adenosine Triphosphate II. CONCERNING THE MECHANISM OF INHIBITION BY THE FRAGMENTED

It has been reported by several groups of workers that incubation of the relaxing factor granar of muscle with magnesium and adenosine triphosphate results in the formation of a soluble relaxing substance capable of inhibiting the magnesium-activated adenosine triphosphatase activity of myofibrils or actomyosin (3-6) and the contraction of single glycerinated muscle fibers (7). In addition, Uriggs and Fuchs have reported that a soluble relaxing substance can be extracted directly from muscle (8) and have suggested that this substance may be a phospholipid (9. The physiological significance of a soluble relaxing substance has been brought into question by experiments (10-14) which have shown that the inhibition by grana or chelating agents of ATPase activity and superprecipitation of actomyosin are accompanied by the removal of bound exchangeable calcium from act.omyosin. We have recently demonstrated a calcium requirement of the magnesium-activated myofibrillar ATPase in the absence of calcium-binding agents (15, 16). Calcium contamination present in commercially available ATP could account for earlier results which suggested that calcium was not required for maximal ATPase activity in the presence of magnesium (17). In vievv of these facts, we have reinvestigated the problem of the soluble relaxing substance and found that the removal of calcium which contaminates commercially available ATP can account for effects previously attributed to a soluble relaxing substance released from relaxing factor grana.