Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells.
Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells.
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艾利希腹水肿瘤细胞磷酸酪氨酰蛋白磷酸酶的部分纯化和表征。
DOI：
10.1021/bi00265a030
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Bornstein,P
中科院分区：
文献类型：
--
作者：
Hörlein,D;Gallis,B;Brautigan,DL;Bornstein,P
Dietrich Horlein, Byron Gallis, David L. Brautigan， and Paul Bornstein*摘要：我们先前在人表皮样癌A431细胞的膜泡中描述了一种磷酸化酪氨酸蛋白磷酸酶，该酶被微摩尔浓度的Zn2+抑制，并且对乙二胺四乙酸（EDTA）和NaF不敏感[Brautigan， D. L., Bornstein， P., & Gallis， B.(1981) J. Biol.]。在这里，我们从埃利希腹水肿瘤细胞的裂解物中鉴定和部分纯化了一种类似的酶。采用二乙基氨基乙基Sephadex、Zn2+亲和层析和Sephadex G-75层析对酶进行纯化。在纯化过程中，磷酸酶被分离成至少三个部分，所有这些部分都表现出非常相似的性质，在凝胶过滤下表观分子量为40000。这种酶能够去磷酸化含有磷酸酪氨酸（P-Tyr）的羧甲基化和琥珀化（CM-SC）磷酸化酶，表观Km为0.8 uM，以及含有P-Tyr的酪蛋白和表皮生长因子（EGF）受体激酶，但不能去磷酸化酪氨酸残基，这是最近发现的蛋白质翻译后修饰（Hunter& Sefton, 1980）。酪氨酸磷酸化水平与某些RNA肿瘤病毒对细胞的转化有关（Sefton等，1980；Barbacid等，1980）。酪氨酸残基特异性蛋白激酶活性首次被证明与劳斯肉瘤病毒的转化蛋白pp60srcl相关（Hunter & Sefton, 1980）。RSV转化的细胞以及其他一些RNA肿瘤病毒（Barbacid et al., 1980; Blomberg et al., 1980; Sefton et al., 1981）显示磷酸化酪氨酸水平升高。当被含有转化基因温度敏感突变的RSV感染的细胞转移到限制性温度进行转化时，60%的磷酸盐从phos-中丢失
Dietrich Horlein, Byron Gallis, David L. Brautigan, and Paul Bornstein* abstract: We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epi-dermoid carcinoma A431 cells which is inhibited by micromolar concentrations of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B.(1981) J. Biol. Chem. 256, 6519-6522], Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. The enzyme was purified by using diethyl-aminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40000 upongel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 uM, as well as P-Tyr-containing casein and epidermal growth factor (EGF) receptor kinase, but did not de-IRiosphorylation of tyrosine residues is a recently discovered posttranslational modification of proteins (Hunter& Sefton, 1980). The level of tyrosine phosphorylation is correlated with the transformation of cells by certain RNA tumor viruses (Sefton et al., 1980; Barbacid et al., 1980). Protein kinase activity specific for tyrosine residues was first shown to be associated with the transforming protein of Rous sarcoma virus, pp60srcl (Hunter & Sefton, 1980). Cells transformed by RSV, as well as some other RNA tumor viruses (Barbacid et al., 1980; Blomberg et al., 1980; Sefton et al., 1981), showed elevated levels of phosphotyrosine. When cells infected by RSV containing a temperature-sensitive mutation in the transforming gene are shifted to the restrictive temperature for transformation, 60% of the phosphate is lost from phos-