Petukhov S P, Lebedeva E L, Bulargina T V, Severin E S
Biokhimiia. 1987 Nov;52(11):1808-17.
A procedure for measuring the activity of tyrosine-specific protein kinases was developed. The method is based on the fact that the phosphoryl groups of phosphotyrosine residues of phosphorylated protein substrates are not hydrolyzed in alkaline solutions, whereas the phosphoserine and phosphothreonine residues lose their phosphoryl groups upon heating in 2 N KOH. It was demonstrated that rat sarcoma XC cells in culture and in solid tumours contain tyrosine-specific protein kinase (TPK). The TPK is localized in the membrane fraction of the cells and is solubilized by 1% Triton X-100. Mn2+-ATP is a nucleotide substrate for TPK. Among other protein substrates, TPK strongly phosphorylates histones H5 and H2a, weakly phosphorylates histones H2b, H4 and H1 but does not phosphorylate protamine, casein, vinculin or angiotensin II. The optimal concentration of Mn2+ for the reaction is 2 mM; the Km values for ATP and histone H5 are 2-3 microM and 2.5 mg/ml, respectively. Tyrosine-specific protein kinases phosphorylating histone H5 were detected in the membrane fraction isolated from different mammalian tissues, e. g., spleen, heart, liver, brain and lungs, as well as from human intestinal mucosa and mucosal adenocarcinoma. These results suggest that tyrosine-specific protein kinases phosphorylating histone H5 are present in a vast majority of mammalian tissues.
开发了一种测量酪氨酸特异性蛋白激酶活性的方法。该方法基于以下事实：磷酸化蛋白底物的磷酸酪氨酸残基的磷酰基在碱性溶液中不会水解，而磷酸丝氨酸和磷酸苏氨酸残基在2N KOH中加热时会失去其磷酰基。已证明，培养的大鼠肉瘤XC细胞和实体瘤中含有酪氨酸特异性蛋白激酶（TPK）。TPK定位于细胞的膜部分，并可被1% Triton X-100溶解。Mn2+-ATP是TPK的核苷酸底物。在其他蛋白底物中，TPK强烈磷酸化组蛋白H5和H2a，弱磷酸化组蛋白H2b、H4和H1，但不磷酸化鱼精蛋白、酪蛋白、纽蛋白或血管紧张素II。该反应的最佳Mn2+浓度为2 mM；ATP和组蛋白H5的Km值分别为2-3 microM和2.5 mg/ml。在从不同哺乳动物组织（如脾脏、心脏、肝脏、大脑和肺）以及人肠黏膜和黏膜腺癌分离的膜部分中检测到磷酸化组蛋白H5的酪氨酸特异性蛋白激酶。这些结果表明，磷酸化组蛋白H5的酪氨酸特异性蛋白激酶存在于绝大多数哺乳动物组织中。
