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 (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent K(m) values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg(2+) was the preferred divalent cation, Mn(2+) could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn(2+) acted synergistically with suboptimal Mg(2+) concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca(2+) and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of (3)H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels.
Westergren
T
Department of Plant Biochemistry, Lund University, Box 117, SE-221 00, Lund, Sweden.
Ekblad
L
Jergil
B
Sommarin
M