Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.
Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.
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鼠 Leydig 肿瘤细胞中蛋白酪氨酸激酶活性的表征。
DOI：
10.1016/0167-4889(86)90136-9
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Melner,MH
中科院分区：
文献类型：
--
作者：
Dangott,LJ;Puett,D;Melner,MH
使用合成肽NH 2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH作为底物，表征来自Leydig肿瘤细胞（M5480 A）的蛋白酪氨酸激酶（EC 2.7.1.37）的活性。在颗粒级分（30 000 × g颗粒）中检测到相对较高的酪氨酸特异性蛋白激酶活性（约135 pmol/mg蛋白/min），并发现其作为时间和蛋白浓度的函数呈线性。通过掺入到肽中的32 PO 4判断，0.02%Nonidet P-40刺激颗粒部分中的酶活性1.4倍。在存在十二烷基硫酸钠的情况下，用[γ-32 P]ATP磷酸化M5480 A颗粒组分中的内源性蛋白质，导致聚丙烯酰胺凝胶中出现几条耐碱放射性标记条带。该组包括表观分子量在50 000-54 000范围内的主要放射性标记双联体。对这些洗脱蛋白质的水解产物进行磷酸氨基酸分析表明存在磷酸酪氨酸。在32 PO 4存在下培养M5480 A细胞后，也检测到几个耐碱性放射性标记带，包括表观分子量为32000的主要双联体。这些研究表明，存在高水平的蛋白质酪氨酸激酶活性的Leydig肿瘤细胞和内源性蛋白质底物的这种酶的活性。
The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 ×gpellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by32PO4incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [γ-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000–54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radiolabeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.