Purification of two immunologically related phosphatidylinositol-(4,5)- bisphosphate phosphatases from bovine brain cytosol.

摘要: Abstract Two phosphatidylinositol-(4,5)-bisphosphate (PtdIns-(4,5)P2) phosphatase activities were isolated from a 45% saturated (NH4)2SO4 fraction of the soluble cytosol (100,000 x g supernatant) bovine cerebral hemispheres by ion-exchange chromatography on Q-Sepharose (Q-1 and Q-2). Each was further purified heparin-Sepharose, butyl-agarose, and/or Cibacron blue F3GA to yield products similar specific activity (70-100 mumol/min/mg protein, 1000-2000-fold purification). Salt required stabilize dithiothreitol preserve maximum prevent or reverse aggregation that resisted disruption mercaptoethanol SDS. Monoclonal antibodies prepared recognized several components in partially preparations. Immunoabsorption monoclonal had been chemically cross-linked protein A-Sepharose followed SDS-polyacrylamide gel electrophoresis absorbed proteins used identify active as 155-kDa Q-1 115-kDa Q-2. different epitopes phosphatase. A third antibody common epitope both phosphatases indicating two enzymes are related. Both Mg(2+)-dependent, exhibited kinetic properties, hydrolyzed PtdIns(4,5)P2 but not PtdIns(4)P, phosphatidic acid, other phosphate monoesters. They inositol (1,4,5)-trisphosphate at 30% rate with this co-purified activity. High molecular weight may be precursors lower Type II polyphosphate-5-phosphatases shown account for platelets (Matzaris, M., Jackson, S.P., Laxminarayan, Speed, C.J., Mitchell, C.A. (1994) J. Biol. Chem. 269, 3397-3402). The three did inhibit native denatured (Western blots) should useful tools study distribution, structure, regulation forms