CHARACTERIZATION OF PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASE ACTIVITIES FROM BOVINE BRAIN MEMBRANES

Phosphatidylinositol-4-phosphate (PtdIns(4)P) kinase activity associated with bovine brain membranes, was released by NaCl treatment and partially purified by chromatography on phosphocellulose, phenylsepharose, Ultrogel AcA44, DEAE-cellulose and ATP-agarose. The final preparation contained a 6333-fold purified protein fraction with a specific activity of 171 nmol . min-1 . mg-1. Under conditions where this PtdIns(4)P kinase activity (PtdIns(4)P kinase activity b) did not bind to DEAE-cellulose, a PtdIns(4)P kinase activity purified earlier (Moritz, A., De Graan, P.N.E., Ekhart, P.F., Gispen, W.H. and Wirtz, K.W.A. (1990) J. Neurochem. 54, 351-354) does bind (PtdIns(4)P kinase activity a). Both enzyme activities specifically used PtdIns(4)P as substrate and phosphorylated the inositol moiety at the 5'-position. PtdIns(4) kinase activity a has an apparent K(m) of 18 muM for PtdIns(4)P whereas PtdIns(4)P kinase activity b has a K(m) of 4 muM. All other measured kinetic parameters (i.e., K(m) for ATP, Mg2+-dependence, pH optimum, activation by phosphatidylserine and inhibition by phosphatidylinositol 4,5-bisphosphate) were similar for both enzyme activities.