Purification and characterization of phosphatidylinositol 4‐kinase from human erythrocyte membranes

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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 [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D‐4 position of the inositol ring. The Km 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.