title: Purification of ras GTPase activating protein from bovine brain.
abstract: In cytosolic extracts of bovine brain, we detected ras GTPase activating protein (GAP) activity that stimulated the GTP hydrolytic activity of normal c-Ha-ras p21 but not that of the oncogenic [Val12]p21 variant. GAP was purified 19,500-fold by a five-column procedure involving DEAE-Sephacel, Sepharose 6B, orange dye and green dye matrices, and Mono Q resins. A single major protein band of 125 kDa was observed on NaDodSO4/polyacrylamide gels that correlated with the elution of GAP activity on Mono Q. Purified GAP was devoid of inherent GTP hydrolytic activity, suggesting that it was a regulator of ras intrinsic GTPase activity. Under submaximal velocity conditions, the second-order rate constant of GTP hydrolysis at 24 degrees C for p21-GTP + GAP (4.5 X 10(6) M-1.sec-1) was at least 1000-fold greater than that for [Val12]p21-GTP + GAP (less than 3 X 10(3) M-1.sec-1).
authors.affiliation: Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
authors.forename: J B
authors.initials: JB
authors.lastname: Gibbs
authors.forename: M D
authors.initials: MD
authors.lastname: Schaber
authors.forename: W J
authors.initials: WJ
authors.lastname: Allard
authors.forename: I S
authors.initials: IS
authors.lastname: Sigal
authors.forename: E M
authors.initials: EM
authors.lastname: Scolnick
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D051381:Rats; D014018:Tissue Distribution; D014981:Xenopus; D020703:ras GTPase-Activating Proteins
chemical_list: D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)