Gibbs J B, Schaber M D, Allard W J, Sigal I S, Scolnick E M
Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Proc Natl Acad Sci U S A. 1988 Jul;85(14):5026-30. doi: 10.1073/pnas.85.14.5026.
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).
在牛脑的胞质提取物中，我们检测到了ras GTP酶激活蛋白（GAP）活性，该活性可刺激正常c-Ha-ras p21的GTP水解活性，但不能刺激致癌性[Val12]p21变体的GTP水解活性。通过涉及DEAE-琼脂糖凝胶、琼脂糖凝胶6B、橙色染料和绿色染料基质以及Mono Q树脂的五柱法，GAP被纯化了19500倍。在NaDodSO4/聚丙烯酰胺凝胶上观察到一条125 kDa的单一主要蛋白带，这与GAP活性在Mono Q上的洗脱相关。纯化后的GAP没有固有的GTP水解活性，这表明它是ras内在GTP酶活性的调节剂。在次最大速度条件下，24℃时p21-GTP + GAP的GTP水解二级速率常数（4.5×10⁶ M⁻¹·sec⁻¹）至少比[Val12]p21-GTP + GAP的二级速率常数（小于3×10³ M⁻¹·sec⁻¹）大1000倍。
