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Regulation Of Phosphatidylinositol Kinases By Arachidonic Acid In Rat Submandibular Gland Cells

H. C. Chung, N. Fleming
Published 2004 · Chemistry, Medicine

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Phosphoinositide kinases were characterized in membrane extracts of rat submandibular gland cells. Both phosphatidylinositol (PI) 4-kinase and phosphatidylinositol-4-phosphate (PI(4)P) 5-kinase phosphorylated endogenous substrates in reactions that were linear for up to 5 min, were activated by Mg2+ and showed maximal activity around neutral pH. PI 4-kinase was stimulated by Triton X-100 at an optimal concentration of 0.22%, but the detergent had an inhibitory effect on PI(4)P 5-kinase. Arachidonic acid (AA), at concentrations greater than 100 μM, inhibited the activity of both enzymes in a dose-dependent manner. The inhibitory effect was replicated by other unsaturated fatty acids, but not by a saturated fatty acid of the sn-20 series. The nature of AA inhibition of the kinases was examined in enzyme kinetic studies with exogenous phosphoinositide and adenosine 5′-triphosphate (ATP) substrates. Lineweaver-Burk plots of PI 4-kinase activity showed that AA had no effect on the apparent Km for either PI or ATP, but that the fatty acid significantly reduced Vmax (PI) from 331 to 177−1.min−1 and Vmax (ATP) from 173 to 59−1.min−1. This inhibitory action was consistent for PI(4)P 5-kinase kinetics, where again, AA did not alter apparent Km values, but lowered Vmax for both PI(4)P and ATP by around 50%. Since the combination of a reduced Vmax and an unchanged Km value indicates noncompetitive enzyme inhibition, it is proposed that AA regulates phosphoinositide cycle activity in submandibular gland cells by acting as a noncompetitive inhibitor of PI 4-kinase and PI(4)P 5-kinase.
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