REGULATION OF PHOSPHATIDYLINOSITOL KINASES BY ARACHIDONIC-ACID IN RATSUBMANDIBULAR-GLAND CELLS

Phosphoinositide kinases were characterized in membrane extracts of ra t submandibular gland cells. Both phosphatidylinositol (PI) 4-kinase a nd phosphatidylinositol-4-phosphate (PI(4)P) 5-kinase phosphorylated e ndogenous substrates in reactions that were linear for up to 5 min, we re activated by Mg2+ and showed maximal activity around neutral pH. PI 4-kinase was stimulated by Triton X-100 at an optimal concentration o f 0.22%, but the detergent had an inhibitory effect on PI(4)P 5-kinase . Arachidonic acid (AA), at concentrations greater than 100 mu M, inhi bited the activity of both enzymes in a dose-dependent manner. The inh ibitory effect was replicated by other unsaturated fatty acids, but no t by a saturated fatty acid of the sn-20 series. The nature of AA inhi bition of the kinases was examined in enzyme kinetic studies with exog enous phosphoinositide and adenosine 5'-triphosphate (ATP) substrates. Lineweaver-Burk plots of PI 4-kinase activity showed that AA had no e ffect on the apparent K-m for either PI or ATP, but that the fatty aci d significantly reduced V-max (PI) from 331 to 177 pmol.mg(-1).min(-1) and V-max (ATP) from 173 to 59 pmol.mg(-1).min(-1). This inhibitory a ction was consistent for PI(4)P 5-kinase kinetics, where again, AA did not alter apparent K-m values, but lowered V-max for both PI(4)P and ATP by around 50%. Since the combination of a reduced V-max and an unc hanged K-m value indicates noncompetitive enzyme inhibition, it is pro posed that AA regulates phosphoinositide cycle activity in submandibul ar gland cells by acting as a noncompetitive inhibitor of PI 4-kinase and PI(4)P 5-kinase.