Phosphatidylinositol 4-kinase associated with spinach plasma membranes. Isolation and characterization of two distinct forms

Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (Ptdlns) kinase activity that was firmly as sociated with the membrane. The enzyme was solubilized by detergent treatme nt (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharos e chromatography. Two enzymically active tractions, QI and QII, both exhibi ting Ptdlns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent K-m value s for ATP (approximately 0.45 mM) and Ptdlns (approximately 0.2 mM) and wer e insensitive to inhibition by adenosine. While Mg2+ was the preferred diva lent cation, Mn2+ could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn2+ acted synergistically with suboptimal Mg2+ concentrations to activate not only the QII enzyme, but al so to some extent QI. Both enzymes were inhibited by millimolar concentrati ons of Ca2+ and micromolar concentrations of wortmannin. The apparent molec ular mass for QI was 120 kD, which was determined by SDS-PACE and western b lotting using an antibody against a peptide unique for lipid kinases and th e binding of H-3-wortmannin, and for QII 65 kD as determined by immunodetec tion and renaturation of Ptdlns kinase activity in the 65-kD region of poly acrylamide gels.