The diphosphoinositide kinase of rat brain

Abstract

1. The supernatant fraction of adult rat brain contains a diphosphoinositide kinase. 2. Formation of triphosphoinositide by the enzyme in the presence of ATP and Mg²⁺ ions was shown with labelled ATP or labelled diphosphoinositide. 3. The kinase was also activated by Ca²⁺, Mn²⁺ and Co²⁺ ions, but to a smaller extent than by Mg²⁺ ions. 4. In the presence of optimum Mg²⁺ ion concentration the enzyme was inhibited by Ca²⁺ ions. 5. Activity did not depend on thiol groups and the pH optimum was 7·3. 6. The dialysed supernatant fraction had no diglyceride kinase activity and negligible phosphatidylinositol kinase activity. 7. Triphosphoinositide phosphomonoesterase was present but showed little activity under the conditions used to assay the kinase. 8. Diphosphoinositide kinase was purified by ammonium sulphate fractionation, ethanol treatment and chromatography on Sephadex G-200. 9. This purification removed much of the triphosphoinositide phosphomonoesterase.