PLANT PHYSIOLOGY , Vol 105, Issue 3 903-909, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Phosphatidate Kinase, A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells)
J. B. Wissing, B. Kornak, A. Funke and B. Riedel
Enzymologie, Gesellschaft fur Biotechnologische Forschung, D-38124 Braunschweig, Germany
Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid
phosphotransferase), a novel enzyme of phospholipid metabolism, was
detected recently in the plasma membranes of suspension-cultured
Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993]
Plant Physiol 102: 1243-1249). In the present work the properties of
phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1
and an isoelectric point of 4.8, and was rather stable in the presence of
its substrates. Although the kinase accepted both ATP and GTP, with Km
values of about 12 and 18 [mu]M, respectively, the only lipid substrate was
phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested
was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate,
the product of the enzyme, it was shown that the kinase catalyzes a
reversible reaction. The activity of the extracted enzyme depended on the
presence of surfactants such as Triton X-100 or [beta]-octylglucoside,
whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton
X-100/phosphatidate mixed micelles performed according to the "surface
dilution" kinetic model showed saturation kinetics with respect to both
bulk and surface concentration of phosphatidate. The interfacial Michaelis
constant for phosphatidate was determined as 0.6 mol %.
