Regulation of phosphatidylinositol kinases by arachidonic acid in rat submandibular gland cells.
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 microM, 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 pmol.mg-1.min-1 and Vmax (ATP) from 173 to 59 pmol.mg-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.
Chung
H C
HC
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Fleming
N
N
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D017830:Octoxynol / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013363:Submandibular Gland / Q000187:drug effects / Q000201:enzymology*
D010710:Phosphates; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D017830:Octoxynol; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D008274:Magnesium