Rübsamen H, Friis R R, Bauer H
Proc Natl Acad Sci U S A. 1979 Feb;76(2):967-71. doi: 10.1073/pnas.76.2.967.
Sera from certain rabbits bearing Schmidt-Ruppin strain Rous sarcoma virus (RSV)-induced tumors precipitated p60(src) from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avain sarcoma virus (ASV)], the molecular weights (M(r)s) ranging from 60,000 to 64,000. The p60(src) immunoprecipitated from cells transformed by each of these strains incorporated [gamma-(32)P]ATP into the M(r) 53,000 subunit of IgG, though with differing activities. No such protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was observed when the following immunoprecipitates were used: from uninfected cells, from untransformed cells infected by Rous-associated virus, or from cells transformed by acute leukosis viruses, avian erythroblastosis virus, or myelocytoma virus 29. The kinase reaction had a pH optimum at pH 5.9 and an apparent K(m) for ATP of 4.9 +/- 2 muM, and was dependent on Mg(2+) (K(b) = 46 +/- 12 mM), for which Ca(2+) was no substitute. The kinase was cyclic AMP independent. In order to test whether the protein kinase reaction is directly catalyzed by p60(src), we compared the in vitro temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the src gene product, the p60(src). Concomitant with the loss of the kinase activity by heat inactivation, p60(src) loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p60(src) kinase is itself dependent on phosphorylation for its activity.
携带施密特 - 鲁平株劳氏肉瘤病毒（RSV）诱导肿瘤的某些兔子的血清，能沉淀来自被同源病毒以及其他毒株[布拉格株RSV、布莱恩高滴度株RSV和禽肉瘤病毒（ASV）的布拉迪斯拉发77株]转化的鸡细胞中的p60(src)，其分子量（M(r)s）在60,000至64,000之间。从这些毒株各自转化的细胞中免疫沉淀的p60(src)，能将[γ-(32)P]ATP掺入IgG的M(r) 53,000亚基中，不过活性有所不同。当使用以下免疫沉淀物时，未观察到这种蛋白激酶活性（ATP：蛋白质磷酸转移酶，EC 2.7.1.37）：来自未感染细胞、来自感染劳氏相关病毒的未转化细胞，或来自被急性白血病病毒、禽成红细胞增多症病毒或骨髓细胞瘤病毒29转化的细胞。激酶反应的最适pH为5.9，ATP的表观K(m)为4.9±2 μM，并且依赖于Mg(2+)（K(b)=46±12 mM），Ca(2+)不能替代Mg(2+)。该激酶不依赖于环磷酸腺苷。为了测试蛋白激酶反应是否直接由p60(src)催化，我们比较了来自感染转化温度敏感突变体和亲本野生型病毒的细胞的激酶活性在体外的温度敏感性。来自突变病毒感染细胞提取物的激酶失活的一级速率常数比来自野生型病毒感染细胞的大2倍。这一结果表明蛋白激酶是src基因产物p60(src)的一种酶活性。随着激酶活性因热失活而丧失时，p60(src)失去其60 - 70%的磷酸含量。去磷酸化的动力学与激酶活性失活的动力学完全平行，表明p60(src)激酶自身的活性依赖于磷酸化作用。
