PURIFICATION OF RAS GTPASE ACTIVATING PROTEIN FROM BOVINE BRAIN
PURIFICATION OF RAS GTPASE ACTIVATING PROTEIN FROM BOVINE BRAIN
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DOI：
10.1073/pnas.85.14.5026
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发表时间：
1988-07-01
影响因子：
11.1
通讯作者：
SCOLNICK, EM
中科院分区：
文献类型：
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作者：
GIBBS, JB;SCHABER, MD;SCOLNICK, EM
在牛脑的细胞质提取物中，我们检测到ras GTPase激活蛋白（GAP）活性，该活性刺激正常c-Ha-ras p21的GTP水解活性，而不刺激致癌[Val12]p21变体的GTP水解活性。GAP通过DEAE-Sephacel、Sepharose 6B、橙色染料和绿色染料基质以及Mono Q树脂的五柱程序纯化19,500倍。在NaDodSO4/聚丙烯酰胺凝胶上观察到一个125 kDa的主要蛋白带，该蛋白带与Mono q上GAP活性的洗脱相关。纯化后的GAP不具有固有的GTP水解活性，这表明它是ras固有GTP酶活性的调节剂。在次最大速度条件下，GTP在24℃时的二阶水解速率常数。C为p21-GTP + GAP的4.5倍。106 M-1 .cntdot。sec-1)比[Val12]p21.sbd至少高出1000倍。GTP + GAP(< 3倍。103 M-1 .cntdot。sec-1)。
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.degree. C for p21-GTP + GAP (4.5 .times. 106 M-1 .cntdot. sec-1) was at least 1000-fold greater than that for [Val12]p21.sbd.GTP + GAP (< 3 .times. 103 M-1 .cntdot. sec-1).