PLANT PHYSIOLOGY , Vol 107, Issue 2 491-500, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells
C. M. Okpodu, W. Gross, W. Burkhart and W. F. Boss
Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-7612 (C.M.O., W.B., W.F.B.)
Previously we reported the presence of a soluble phosphatidylinositol
4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture
cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We
have purified the enzyme over 1000-fold using Q-Sepharose ion exchange,
hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of
the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase
activity was recovered after renaturation of the 80-kD region of
polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to
the yeast 55-kD membrane-associated PI 4-kinase on western blots. The
isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or
phosphatidylinositol monophosphate. Maximal PI kinase activity occurred
when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The
enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was
more effective than Mg2+ in increasing enzyme activity; however, maximal
activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had
no effect on the enzyme activity. The Km of the enzyme for ATP was
estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by
adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the
activity. The biochemical characteristics of the carrot soluble PI 4-kinase
are compared with the previously reported PI 4-kinases from animals and
yeast.
