PURIFICATION OF 2 IMMUNOLOGICALLY RELATED PHOSPHATIDYLINOSITOL-(4,5)-BISPHOSPHATE PHOSPHATASES FROM BOVINE BRAIN CYTOSOL

Two phosphatidylinositol-(4,5)-bisphosphate (PtdIns(4,5)P2) phosphatas e activities were isolated from a 45% saturated (NH4)2SO4 fraction of the soluble cytosol (100,000 x g supernatant) of bovine cerebral 'hemi spheres by ion-exchange chromatography on Q-Sepharose (Q-1 and Q-2). E ach was further purified on heparin-Sepharose, butyl-agarose, and/or C ibacron blue F3GA to yield products of similar specific activity (70-1 00 mumol/min/mg protein, 1000-2000-fold purification). Salt was requir ed to stabilize activity and dithiothreitol was required to preserve m aximum activity and to prevent or reverse aggregation that resisted di sruption by mercaptoethanol and/or SDS. Monoclonal antibodies were pre pared that recognized several components in the partially purified pre parations. Immunoabsorption of activity by monoclonal antibodies that had been chemically cross-linked to protein A-Sepharose followed by SD S-polyacrylamide gel electrophoresis of absorbed proteins was used to identify the active components as a 155-kDa protein in Q-1 and a 115-k Da protein in Q-2. Two antibodies recognized different epitopes in the 155-kDa phosphatase. A third antibody recognized a common epitope in both phosphatases indicating that the two enzymes are related. Both ph osphatases were Me2+-dependent, exhibited similar kinetic properties, and hydrolyzed PtdIns(4,5)P2 but not PtdIns(4)P, phosphatidic acid, or several other phosphate monoesters. They hydrolyzed inositol (1,4,5)- trisphosphate at 30% of the rate with PtdIns(4,5)P2 and this activity co-purified with PtdIns(4,5)P2 phosphatase activity. High molecular we ight PtdIns(4,5)P2 phosphatases may be precursors of lower molecular w eight soluble Type II inositol polyphosphate-5-phosphatases shown to a ccount for the PtdIns(4,5)P2 phosphatase activity in platelets (Matzar is, M., Jackson, S. P., Laxminarayan, M., Speed, C. J., and Mitchell, C. A. (1994) J. Biol. Chem. 269,3397-3402). The three antibodies did n ot inhibit activity but recognized both native and denatured (Western blots) phosphatases and should be useful tools to study the distributi on, structure, and regulation of the two forms of PtdIns(4,5)P2 phosph atase.