Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.