CHARACTERIZATION OF THE P21 RAS ONCOGENE PRODUCT

负责肿瘤发生的v-ras-H致癌基因已被证实

The v-ras-H oncogene, which is responsible for tumorigenesis, has been cloned into E. coli expression vector, and its gene product was purified in a large amount which is sufficient for biochemical and enzymological analyses. Purified protein showed full biochemical activities of GTP/GDP binding, autokinase and GTPase. In order to determine the active site of p21, the effects of antibodies against p21 and N-ethylmaleimide (NEM) were studied. Among six monoclonals, only Y13-259 inhibited p21 activities as much as 80%, which indicated that Y13-259 affected the active site. NEM inactivated p21 activities, indicating that cysteine residue is involved in the active site. The active site could also be specifically labeled by a photoaffinity analogue, P3-(4-azidoanilido)-5'GTP. It was found that monoclonal antibodies against p21 weakly cross-immunoprecipitated the G-protein of adenylate cyclase labeled by 32P-ADP-ribosylation, suggesting that either there might be weak interaction between p21 and G-protein or there might be structural homology between two proteins. New monoclonal antibodies were obtained after immunization of p21 expressed in E. coli into mice.