Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.