Two species of Ptdlns 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-I 00, 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 ["PJPtdInsP products formed by the purified Ptdlns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring.The K, 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.Stimulated turnover of inositol phospholipids is intimately involved in the response of cells to a wide variety of hormones, growth factors and neurotransmitters [I]. A great deal of attention has been focused on the rapid receptor-mediated hydrolysis of Ptdlns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) by phospholipase C in many cell types. The products of this reaction, inositol trisphosphate and diacylglycerol, serve as messenger molecules by mobilizing intracellular Ca2+ and activating protein kinase C, respectively [2, 31.Recent findings suggest that the production of both Ptdlns4P (phosphatidylinosito14-phosphate) and PtdIns(4,5) P2 by sequential phosphorylation of PtdIns via PtdIns4P may also be regulated by extracellular agonists [4, 51. In addition to providing substrates for intracellular messenger production, altered levels of PtdIns4P and PtdIns(4,5)P2 may be of significance for Ca2 + homeostasis [6] and actin polymerization [7]. However the transducing mechanism of agonist-dependent PtdIns 4-phosphorylation is not yet understood. Several recent results point to a regulation of PtdIns 4-kinase activity by protein kinase C or by protein-tyrosine-kinase-catalyzed phosphorylation [4,8 -lo], but the evidence for this is circumstantial.The complexity and physiological importance of the various PtdIns-phosphorylating reactions are underlined by the Correspondence to R. Wetzker, Institut fur Biochemie, Lobderstrasse 3, 0-6900 Jena, Federal Republic of Germany Ahhrcviutions. InslP, inositol 1-phosphate; Ins4P, inositol 4-phosphate; PtdIns 3P, phosphatidylinositol 3-phosphate; PtdIns 4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol4,5-bisphosphate.Enzytne. PtdIns kinase (EC 2.7.1.67).