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PURIFICATION AND CHARACTERIZATION OF PHOSPHATIDYLINOSITOL 4-KINASE FROM HUMAN ERYTHROCYTE-MEMBRANES

WETZKER, R; KLINGER, R; HSUAN, J; FRY, MJ; KAUFFMANNZEH, A; MULLER, E; ... WATERFIELD, M; + view all (1991) PURIFICATION AND CHARACTERIZATION OF PHOSPHATIDYLINOSITOL 4-KINASE FROM HUMAN ERYTHROCYTE-MEMBRANES. EUR J BIOCHEM , 200 (1) 179 - 185.

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Abstract

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes.Characterization of the [P-32]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring.The K(m) values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.

Type:Article
Title:PURIFICATION AND CHARACTERIZATION OF PHOSPHATIDYLINOSITOL 4-KINASE FROM HUMAN ERYTHROCYTE-MEMBRANES
Keywords:HUMAN PLATELETS, KINASE, CALCIUM, BRAIN, TURNOVER, POLYPHOSPHOINOSITIDES, STIMULATION, ACTIVATION, RECEPTORS, CA-2+

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