Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase.
Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D008297:Male; D008871:Microvilli / Q000201:enzymology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010726:Phosphodiesterase Inhibitors; D010740:Phospholipases / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D002413:Cations, Divalent; D010726:Phosphodiesterase Inhibitors; D003840:Deoxycholic Acid; D000255:Adenosine Triphosphate; D010740:Phospholipases; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase