Purification of the major protein-tyrosine-phosphatases of human placenta.
Purification of the major protein-tyrosine-phosphatases of human placenta.
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DOI：
10.1016/s0021-9258(18)68702-2
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发表时间：
1988-05
期刊：
影响因子：
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通讯作者：
N. Tonks;C. D. Diltz;E. Fischer
中科院分区：
文献类型：
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作者：
N. Tonks;C. D. Diltz;E. Fischer
该报告描述了从人胎盘中纯化主要蛋白酪氨酸磷酸酶的情况。采用新型人工底物（即还原的羧酰胺甲基化和马来酰化溶菌酶）跟踪酶活性，该酶通过同样来自人胎盘的胰岛素和表皮生长因子受体激酶的部分纯化制剂在酪氨酸上进行磷酸化。蛋白质酪氨酸磷酸酶纯化的关键步骤是在硫代磷酸化、还原、羧酰胺甲基化和马来酰化溶菌酶-琼脂糖柱上进行亲和层析。与可溶性和颗粒级分分开进行纯化。尽管从每种酶中获得了多种不同的酶形式，但“可溶性”酶亚型与其“颗粒”对应物的行为之间几乎没有检测到差异。主要亚型被纯化至明显均质，可溶性部分富集约 23,000 倍，产率 10%，颗粒部分富集 4,300 倍，产率 13%。两个样品在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上均迁移为 35 kDa 条带，释放的 Pi min-1 mg-1 的比活性约为 45,000 nmol，至少比 1 型和 2A 型丝氨酸/苏氨酸磷酸酶高 2-3 倍。人胎盘可溶性部分中的蛋白质酪氨酸磷酸酶水平（2,000 单位/克蛋白质）与骨骼肌中的蛋白质丝氨酸/苏氨酸磷酸酶 1 和 2A 大致相同。
This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the “soluble” enzyme subtypes and their “particulate” counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle.