Yang X M, Walter G
Department of Pathology, University of California, San Diego, La Jolla 92093.
Oncogene. 1988 Sep;3(3):237-44.
Several tumor lines of neuronal origin were assayed for the presence of two forms of pp60c-src, designated pp60 and pp60+. To determine the specific kinase activity of pp60 and pp60+, an enzymatic analysis was carried out with neuroblastoma LA-N-5, containing a high level of pp60+, neuroblastoma SK-N-SH, containing a low level of pp60+, fibroblast FSD, containing pp60 but no pp60+, and Rous sarcoma virus transformed 3T3 cells, SR-3T3, containing pp60v-src. Km values for Mg2+-ATP of approximately 21, 8, 17 and 13 microM were determined for pp60src kinase from LA-N-5, SK-N-SH, FSD and SR-3T3 respectively, using enolase as a substrate. The Vmax values for pp60c-src kinase from LA-N-5, SK-N-SH and FSD were similar. The Vmax value of pp60v-src was about 50-fold higher. Thirteen distinct phosphopeptides were found in tryptic digests from pp60 and pp60+ labeled in vivo with [32P]. The presence of one phosphopeptide, derived from the N-terminus, correlated with the level of pp60+ in neuroblastoma cells, but a small amount of this peptide was also detected in fibroblasts. Only phosphoserine was detected in the N-terminus of pp60 and pp60+. Our data strongly suggest that the six extra amino acids, present in pp60+ but not in pp60, do not significantly alter the specific kinase activity of pp60+, but might influence its phosphorylation state.
检测了几种神经源性肿瘤细胞系中两种形式的pp60c-src（分别命名为pp60和pp60+）的存在情况。为了确定pp60和pp60+的特异性激酶活性，对含有高水平pp60+的神经母细胞瘤LA-N-5、含有低水平pp60+的神经母细胞瘤SK-N-SH、含有pp60但不含pp60+的成纤维细胞FSD以及含有pp60v-src的劳氏肉瘤病毒转化的3T3细胞SR-3T3进行了酶活性分析。以烯醇酶为底物，分别测定了来自LA-N-5、SK-N-SH、FSD和SR-3T3的pp60src激酶对Mg2+-ATP的Km值，分别约为21、8、17和13微摩尔。来自LA-N-5、SK-N-SH和FSD的pp60c-src激酶的Vmax值相似。pp60v-src的Vmax值约高50倍。在用[32P]体内标记的pp60和pp60+的胰蛋白酶消化物中发现了13种不同磷酸肽。一种源自N端的磷酸肽的存在与神经母细胞瘤细胞中pp60+的水平相关，但在成纤维细胞中也检测到少量这种肽。在pp60和pp60+的N端仅检测到磷酸丝氨酸。我们的数据有力地表明，pp60+中存在但pp60中不存在的六个额外氨基酸不会显著改变pp60+的特异性激酶活性，但可能会影响其磷酸化状态。
