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 x 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 Km of 18 microM for PtdIns(4)P whereas PtdIns(4)P kinase activity b has a Km of 4 microM. All other measured kinetic parameters (i.e., Km for ATP, Mg(2+)-dependence, pH optimum, activation by phosphatidylserine and inhibition by phosphatidylinositol 4,5-bisphosphate) were similar for both enzyme activities.
Center for Biomembranes and Lipid Enzymology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
A
A
Moritz
J
J
Westerman
P N
PN
De Graan
B
B
Payrastre
W H
WH
Gispen
K W
KW
Wirtz
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D002853:Chromatography, Liquid; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity
D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase