Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes

Abstract

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[γ- 32P]ATP. The K m 's for 32P incorporation into di- and triphosphoinositide were 110 and 48 μ m ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 °C, whereas the optimum for triphosphoinositide labeling occurred at 43 °C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 °C but not at 25 °C 32P-labeled diphosphoinositide was phosphorylated to triphosphoinositide in the presence of Mg-ATP. Triton X-100 partially inhibited 32P incorporation into diphosphoinositide but completely inhibited the synthesis of triphosphoinositide. At physiological concentrations, 0.4 m m MgCl 2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl 2 (5 to 50 m m) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (⩽100 m m) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 m m NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 m m MgCl 2 and 200 m m NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 m m MgCl 2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.