Open AccessPurification and characterization of phosphatidylinositol 4‐kinase from human erythrocyte membranes
Author(s) -
WETZKER Reinhard,
KLINGER Reinhard,
HSUAN Justin,
FRY Michael J.,
KAUFFMANNZEH Andrea,
MÜLLER Elke,
FRUNDER Horst,
WATERFIELD Michael
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb21065.x
Subject(s) - phosphatidylinositol , membrane , kinase , characterization (materials science) , erythrocyte membrane , microbiology and biotechnology , chemistry , biochemistry , biology , materials science , nanotechnology
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 [ 32 P]PtdIns P 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 Mg 2+ . and inhibited by Ca 2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.