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. 1988 Jul;85(14):5026-30.
doi: 10.1073/pnas.85.14.5026.

Purification of ras GTPase activating protein from bovine brain

J B Gibbs  1 , M D Schaber, W J Allard, I S Sigal, E M Scolnick
Affiliations

Purification of ras GTPase activating protein from bovine brain

J B Gibbs et al. Proc Natl Acad Sci U S A. 1988 Jul.

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).

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