Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody
| Autor: | Thomas Y. Shih, S Hattori, K Halliday, L S Ulsh |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
GTP'
medicine.drug_class GTPase Oncogene Protein p21(ras) Protein Serine-Threonine Kinases Biology Antibodies Viral Monoclonal antibody Guanosine Diphosphate GTP Phosphohydrolases Mice chemistry.chemical_compound Escherichia coli medicine Animals Cloning Molecular Binding site Molecular Biology Cells Cultured Binding Sites GDP binding Antibodies Monoclonal Cell Biology Fusion protein Molecular biology Neoplasm Proteins Biochemistry chemistry Guanosine diphosphate Harvey murine sarcoma virus Guanosine Triphosphate Protein Kinases Research Article |
| Zdroj: | Molecular and Cellular Biology. 5:1449-1455 |
| ISSN: | 1098-5549 0270-7306 |
| DOI: | 10.1128/mcb.5.6.1449 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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