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.