Tung H Y, Reed L J
Anal Biochem. 1987 Mar;161(2):412-9. doi: 10.1016/0003-2697(87)90469-6.
A protein tyrosine kinase with an apparent Mr of 60,000 was highly purified from bovine spleen and used to phosphorylate poly(Glu, Tyr) (4:1) on tyrosine residues for the study of phosphotyrosyl protein phosphatases from this tissue. About 70% of the phosphotyrosyl protein phosphatase activity in extracts of bovine spleen was adsorbed on DEAE-Sepharose. Chromatography of the eluted phosphotyrosyl protein phosphatases on phosphocellulose indicated the presence of at least two species, one that did not bind to the phosphocellulose and a second species that did bind and was eluted at about 0.5 M NaCl. The phosphatase that did not bind to phosphocellulose was further purified by successive chromatography on poly(L-lysine)-Sepharose, L-tyrosine-agarose, poly(Glu,Tyr)-Sepharose, and Sephacryl S-200. The enzyme had an apparent Mr of 50,000 as estimated by gel filtration and 52,000 as estimated by NaDodSO4- polyacrylamide gel electrophoresis. The phosphatase exhibited a pH optimum of 6.5-7.0, was inhibited by Zn2+ and vanadate ions, and was stimulated by EDTA. Sodium fluoride and sodium pyrophosphate, inhibitors of phosphoseryl protein phosphatases, had no effect on the enzyme. Protein inhibitors of type 1 phosphoseryl/threonyl phosphatase were also ineffective.
从牛脾脏中高度纯化出一种表观分子量为60,000的蛋白质酪氨酸激酶，用于使聚（谷氨酸，酪氨酸）（4:1）的酪氨酸残基磷酸化，以研究该组织中的磷酸酪氨酸蛋白磷酸酶。牛脾脏提取物中约70%的磷酸酪氨酸蛋白磷酸酶活性吸附在DEAE-琼脂糖上。在磷酸纤维素上对洗脱的磷酸酪氨酸蛋白磷酸酶进行色谱分析表明存在至少两种物质，一种不与磷酸纤维素结合，另一种与磷酸纤维素结合并在约0.5M NaCl浓度下洗脱。不与磷酸纤维素结合的磷酸酶通过在聚（L-赖氨酸）-琼脂糖、L-酪氨酸-琼脂糖、聚（谷氨酸，酪氨酸）-琼脂糖和Sephacryl S-200上连续色谱进一步纯化。通过凝胶过滤估计该酶的表观分子量为50,000，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计为52,000。该磷酸酶的最适pH为6.5 - 7.0，受Zn2+和钒酸盐离子抑制，受EDTA刺激。磷酸丝氨酸蛋白磷酸酶的抑制剂氟化钠和焦磷酸钠对该酶无作用。1型磷酸丝氨酸/苏氨酸磷酸酶的蛋白质抑制剂也无效。
