Müller O, Frech M, Gideon P, Wittinghofer A, Schwarz M
German Cancer Research Center, Project Group Tumor Promotion in the Liver, Heidelberg, Germany.
Oncogene. 1992 Jul;7(7):1407-12.
The intrinsic GTPase activity of the cellular protein p21ras is strongly increased by two cytosolic proteins, the GTPase-activating protein (GAP) produced by the neurofibromatosis type 1 gene (NF1-GAP) and a GAP of 120 kDa molecular mass (p120-GAP). The GAP-mediated stimulation of p21ras GTPase activity was measured in cytosol obtained from carcinogen-induced liver tumors and normal liver tissues of mice of two strains, namely C3H/He and C57BL/6J. For this purpose, cytosolic extracts were incubated with recombinant human p21ras complexed to [gamma-32P]GTP and the time-dependent decrease in p21ras bound radioactivity was measured. Liver cytosolic extracts mediated an increase in the GTPase activity of wild-type p21ras. There were great differences between tumor and normal tissues in the maximal velocity (Vmax) and in the apparent Michaelis constant (KM) of the p21ras GTPase reaction. Both Vmax and apparent KM were decreased in the liver tumors. Cytosolic extracts isolated from liver tumors that harbored point mutations in codon 61 of the c-H-ras gene did not differ in their activity from extracts obtained from non-mutated liver tumors. Since both GAP proteins are important cellular regulators of the ras signaling pathway and probably also effectors of p21ras, the observed differences in GAP activity may be of relevance for the tumorigenic process in mouse liver.
细胞蛋白p21ras的内在GTP酶活性被两种胞质蛋白显著增强，这两种蛋白分别是由1型神经纤维瘤病基因产生的GTP酶激活蛋白（NF1-GAP）和一种分子量为120 kDa的GAP（p120-GAP）。在从两种品系（即C3H/He和C57BL/6J）小鼠的致癌物诱导肝肿瘤和正常肝组织中获得的胞质溶胶中，测量了GAP介导的p21ras GTP酶活性刺激作用。为此，将胞质提取物与与[γ-32P]GTP复合的重组人p21ras一起孵育，并测量p21ras结合放射性随时间的下降情况。肝胞质提取物介导了野生型p21ras GTP酶活性的增加。在p21ras GTP酶反应的最大速度（Vmax）和表观米氏常数（KM）方面，肿瘤组织和正常组织之间存在很大差异。肝肿瘤中的Vmax和表观KM均降低。从c-H-ras基因第61密码子发生点突变的肝肿瘤中分离出的胞质提取物，其活性与从未发生突变的肝肿瘤中获得的提取物没有差异。由于这两种GAP蛋白都是ras信号通路重要的细胞调节因子，可能也是p21ras的效应器，因此观察到的GAP活性差异可能与小鼠肝脏的致瘤过程有关。
