Synthesis of diphosphoinositide by a supernatant fraction from Acanthamoeba castellanii

Publisher Website
Google Scholar
Abstract
Homogenates of the free-living amoeba A. castellanii incorporate phosphate from [.gamma.-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on 1- and 2-dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100,000 g. Incorporation of phosphate from [.gamma.-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ or Mn2+ as well as ATP, and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol is 2 mM. Both ADP and c[cyclic]AMP inhibit the reaction.