Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.
The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.
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Halliday
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006251:Harvey murine sarcoma virus / Q000276:immunology; D051379:Mice; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism*; D015689:Oncogene Protein p21(ras); D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D009363:Neoplasm Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases; D015689:Oncogene Protein p21(ras)