Substrate Regulation of the Sarcoplasmic Reticulum ATPase

The rate of phosphorylation of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles by ITP and ATP was studied using a millisecond mixing and quenching device. The rate of phosphorylation was slower when the vesicles were preincubated in a Ca2+free medium than when preincubated with Ca’+, regardless of the substrate used and of the pH of the medium. When the vesicles were preincubated with Ca2’ at pH 7.4 an overshoot of phosphorylation was observed in the presence of ITP. The overshoot was abolished when the pH of the medium was decreased to 6.0 or when the vesicles were preincubated in a Ca2+free medium. Using vesicles preincubated with Ca2+ the apparent K,,, for ITP found was 2.5 mM at pH 6.0 and 1.0 mu at pH 7.4. The V,,,, observed (77 pmol g-’ s-l) did not change with the pH of the medium. Both at pH 6.0 and 7.4 the apparent K,,, for ATP was 3 PM when the vesicles were preincubated with Ca2+ and 50 PM when preincubated in a Ca’+-free medium. At pH 6.0 the I’,,,,, for ATP varied from 96 to 33 pmol g-’ s-’ depending on whether the vesicles were preincubated in the presence or absence of Ca’+. At pH 7.4 the V,,, for ATP was 90 pm01 g-’ s-l in both conditions. The rate of phosphorylation of the vesicles was dependent on the relative Ca2+ and Mg2’ concentrations of the reaction medium regardless of the substrate used.