Purification of two immunologically related phosphatidylinositol-(4,5)- bisphosphate phosphatases from bovine brain cytosol.
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) of bovine cerebral hemispheres by ion-exchange chromatography on Q-Sepharose (Q-1 and Q-2). Each was further purified on heparin-Sepharose, butyl-agarose, and/or Cibacron blue F3GA to yield products of similar specific activity (70-100 mumol/min/mg protein, 1000-2000-fold purification). Salt was required to stabilize activity and dithiothreitol was required to preserve maximum activity and to prevent or reverse aggregation that resisted disruption by mercaptoethanol and/or SDS. Monoclonal antibodies were prepared that recognized several components in the partially purified preparations. Immunoabsorption of activity by monoclonal antibodies that had been chemically cross-linked to protein A-Sepharose followed by SDS-polyacrylamide gel electrophoresis of absorbed proteins was used to identify the active components as a 155-kDa protein in Q-1 and a 115-kDa 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 phosphatases were Mg(2+)-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 weight PtdIns(4,5)P2 phosphatases may be precursors of lower molecular weight soluble Type II inositol polyphosphate-5-phosphatases shown to account for the PtdIns(4,5)P2 phosphatase activity in platelets (Matzaris, M., Jackson, S.P., Laxminarayan, M., Speed, C.J., and Mitchell, C.A. (1994) J. Biol. Chem. 269, 3397-3402). The three antibodies did not inhibit activity but recognized both native and denatured (Western blots) phosphatases and should be useful tools to study the distribution, structure, and regulation of the two forms of PtdIns(4,5)P2 phosphatase.
Palmer
F B
FB
Atlantic Research Centre, Halifax, Nova Scotia.
Théolis
R
R
Cook
H W
HW
Byers
D M
DM
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000099182:Phosphoinositide 5-Phosphatases
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D010744:Phosphoric Monoester Hydrolases; D000099182:Phosphoinositide 5-Phosphatases