Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.
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.
Wetzker
R
R
Institut für Biochemie, Jena, Federal Republic of Germany.
Klinger
R
R
Hsuan
J
J
Fry
M J
MJ
Kauffmann-Zeh
A
A
Müller
E
E
Frunder
H
H
Waterfield
M
M
D019870:1-Phosphatidylinositol 4-Kinase; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase