Purification of ras GTPase activating protein from bovine brain.
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).
Gibbs
J B
JB
Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Schaber
M D
MD
Allard
W J
WJ
Sigal
I S
IS
Scolnick
E M
EM
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
D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)