Plant Physiol, October 1999, Vol. 121, pp. 507-516
Phosphatidylinositol 4-Kinase Associated with Spinach Plasma
Membranes. Isolation and Characterization of Two Distinct
Forms1
Tomas
Westergren,
Lars
Ekblad,
Bengt
Jergil, and
Marianne
Sommarin*
Department of Plant Biochemistry, Lund University, Box 117, SE-221
00, Lund, Sweden (T.W., M.S.); and Department of Biochemistry, Centre
for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00, Lund, Sweden (L.E., B.J.)
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 Km values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were
insensitive to inhibition by adenosine. While Mg2+ was the
preferred divalent 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 also to some extent QI. Both enzymes were inhibited by millimolar
concentrations of Ca2+ 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
3H-wortmannin, and for QII 65 kD as determined by
immunodetection and renaturation of PtdIns kinase activity in the 65-kD
region of polyacrylamide gels.
1
This work was supported by the Swedish Council
for Forestry and Agricultural Research, the Swedish Natural Science
Research Council, and the Swedish Foundation for Strategic Research.
*
Corresponding author; e-mail marianne.sommarin{at}plantbio.lu.se;
fax 46-46-2224116.
© 1999 American Society of Plant Physiologists
