Daum G, Zander N F, Morse B, Hurwitz D, Schlessinger J, Fischer E H
Department of Biochemistry SJ-70, University of Washington, Seattle 98195.
J Biol Chem. 1991 Jul 5;266(19):12211-5.
The receptor-linked tyrosine phosphatase RPTP alpha from human brain (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R., Ravera, M., Ricca, G., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7000-7004) was expressed in insect cells following infection with recombinant baculovirus. Two major forms of the enzyme, with molecular sizes of 98 kDa and 114 kDa, were detected by immunoblot analysis. This heterogeneity could be ascribed to N-linked glycosylation on the basis of two lines of evidence; namely, blockage of glycosylation with tunicamycin in vivo and removal of carbohydrates by endoglycosidase F in vitro. The 114-kDa form was purified to homogeneity by chromatography on Superose 12 and Mono Q. Compared to the low Mr placenta and T-cell tyrosine phosphatases, RPTP alpha displayed a low optimum pH of 6 and a high Km in the micromolar range toward two artificial substrates (tyrosyl-phosphorylated myelin basic protein and modified lysozyme, respectively). Most effectors had a different and often an opposite influence on phosphatase activity depending on the nature of the substrate and the pH at which the assays were performed. Determination of Km and Vmax values for RPTP alpha suggests that the enzyme could exist in low and high substrate affinity states.
人脑来源的受体连接型酪氨酸磷酸酶RPTPα（卡普兰，R.，莫尔斯，B.，休布纳，K.，克罗齐，C.，豪克，R.，拉韦拉，M.，里卡，G.，杰伊，M.，和施莱辛格，J.（1990年）《美国国家科学院院刊》87卷，7000 - 7004页）在感染重组杆状病毒后在昆虫细胞中表达。通过免疫印迹分析检测到该酶的两种主要形式，分子量分别为98 kDa和114 kDa。基于两条证据线索，这种异质性可归因于N - 连接糖基化；即体内用衣霉素阻断糖基化以及体外用内切糖苷酶F去除碳水化合物。通过Superose 12和Mono Q柱层析将114 kDa形式的酶纯化至均一。与低分子量的胎盘和T细胞酪氨酸磷酸酶相比，RPTPα对两种人工底物（分别为酪氨酸磷酸化的髓鞘碱性蛋白和修饰的溶菌酶）表现出较低的最适pH值6以及微摩尔范围内的高Km值。大多数效应物根据底物的性质和进行测定的pH值对磷酸酶活性有不同且往往相反的影响。对RPTPα的Km和Vmax值的测定表明该酶可能以低底物亲和力状态和高底物亲和力状态存在。
