Bolen J B, Israel M A
J Biol Chem. 1983 Dec 25;258(24):15135-40.
The middle T antigen (MT Ag) encoded by polyoma virus has an associated protein kinase activity which transfers a phosphoryl group from ATP or GTP to a tyrosine residue on MT Ag in immunoprecipitates formed between polyoma virus-infected or transformed cell extracts and serum from animals bearing polyoma-induced tumors. Incubation of such immunoprecipitates or polyoma-transformed cell extracts prior to immunoprecipitation with the sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant inhibition of MT Ag-associated kinase activity. Inactivation of this enzyme activity by NEM was found to be dependent upon the incubation pH, time of incubation, and NEM concentration. ATP, GTP, and ADP in the presence of Mg2+ were found to decrease the rate of NEM-mediated inactivation of MT Ag-associated kinase activity, while CTP and UTP did not detectably alter the rate of enzyme inhibition by NEM. These results suggest that the MT Ag-associated kinase possesses at least one NEM-sensitive sulfhydryl group essential for phosphotransferase activity which may be present at or near the enzyme catalytic site.
多瘤病毒编码的中间T抗原（MT Ag）具有相关的蛋白激酶活性，该活性可将磷酸基团从ATP或GTP转移至MT Ag上的酪氨酸残基，此反应发生在多瘤病毒感染或转化的细胞提取物与携带多瘤病毒诱导肿瘤的动物血清形成的免疫沉淀物中。在用巯基试剂N-乙基马来酰亚胺（NEM）进行免疫沉淀之前，将此类免疫沉淀物或多瘤病毒转化的细胞提取物进行孵育，会导致MT Ag相关激酶活性受到显著抑制。发现NEM对该酶活性的失活取决于孵育pH、孵育时间和NEM浓度。在Mg2+存在的情况下，ATP、GTP和ADP可降低NEM介导的MT Ag相关激酶活性失活速率，而CTP和UTP未检测到对NEM抑制酶活性速率的明显改变。这些结果表明，MT Ag相关激酶拥有至少一个对磷转移酶活性至关重要的NEM敏感巯基，该巯基可能存在于酶催化位点处或附近。
