ATPASE AND PHOSPHATIDYLINOSITOL KINASE ACTIVITIES OF ADRENAL CHROMAFFIN VESICLES

— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg 2+ or Mn 2+ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg 2+ while maximal PI kinase activity required 100 mM Mg 2+ Similar differences were observed with Mn 2+ with the exception that maximal ATPase activity occurred with 0.5 mM Mn 2+ and maximal PI kinase activity occurred with 5 mM Mn 2+ Mn 2+ was more effective than Mg 2+ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg 2+ was 5‐fold greater than the maximal activity obtained with Mn 2+ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins. The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total 32 P‐incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.