The tyrosyl kinase and phosphatidylinositol (PI) kinase activities of human red cells have been partially purified and characterized. Although the PI kinase required detergent for solubilization, the major tyrosyl kinase of the red cell could be extracted by high salt. A very small residual activity remained associated with the membranes, however, that was solubilized with the PI kinase and copurified through an ammonium sulfate precipitation and diethylaminoethyl (DEAE) ion‐exchange step gradient elution. However, the two activities were found to differ with respect to their apparent Kms for ATP and Mg2+; they showed different half‐lives for temperature inactivation, possessed different relative activities in the presence of Mn+ and Ca2+, and were separable by elution from a DEAE‐Trisacryl ion exchange column using a linear NaCl gradient. The kinetic parameters of the membrane‐associated tyrosyl kinase differed from those of the salt‐extracted enzyme. PI kinase was not activated by pretreatment with the tyrosyl kinase p68v‐ros or by addition of the phosphotyrosyl phosphatase inhibitor, vanadate, to intact membranes, and was not competitively inhibited by the tyrosyl kinase substrate poly(Glu4,Tyr). We conclude that the human red cell phosphatidylinositol and tyrosyl kinases are distinct and separable activities, and that at least two separable tyrosyl kinases are present in human erythrocytes.

The interaction of the dissociated catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (kinase A) and phosphatidylinositol metabolism has been studied in rat spleen lymphocyte membranes. As reported previously (Sarkadi et al., FEBS Lett. 152: 195-198, 1983) addition of kinase A increased by about twofold the phosphorylation of phosphatidylinositol in lymphocyte membranes at low ATP concentrations. However, we have found that this increase is an artifact of the assay conditions, and that the increase is a consequence of an inhibition of membrane adenosine triphosphatase activity by the protein kinase A. When lipid phosphorylation was measured under initial rate conditions, at high ATP concentrations, the increase was abolished. No effect of kinase A was observed on initial rates of the synthesis or hydrolysis of phosphatidylinositol phosphate. No phosphatidylinositol bisphosphate was produced in the membranes under any of the assay conditions used.