Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes.
Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[gamma-32P]ATP. The Km's for 32P incorporation into di- and triphosphoinositide were 110 and 48 microM ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 degrees C, whereas the optimum for triphosphoinositide labeling occurred at 43 degrees C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 degrees C but not at 25 degrees 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 mM MgCl2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl2 (5 to 50 mM) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (less than or equal to 100 mM) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 mM NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 mM MgCl2 and 200 mM NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 mM MgCl2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017830:Octoxynol; D010770:Phosphotransferases / Q000097:blood* / Q000302:isolation & purification; D011092:Polyethylene Glycols; D011817:Rabbits; D012492:Salts; D013696:Temperature
D012492:Salts; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase