Zhang Z, Zhao S, Long F, Zhang L, Bai G, Shima H, Nagao M, Lee E Y
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
J Biol Chem. 1994 Jun 24;269(25):16997-7000.
A chimeric mutant was constructed in which a 4-amino acid region (GEFD, residues 274-277) of rabbit muscle protein phosphatase-1 was replaced with the sequence YRCG corresponding to residues 267-270 of rabbit protein phosphatase-2A. This was based on the findings of a gene mutation in okadaic acid-resistant cells which results in a Cys-->Gly conversion in protein phosphatase-2A. The YRCG mutant of protein phosphatase-1 was expressed and purified. The properties of the mutant enzyme were investigated in terms of its sensitivity toward several toxin inhibitors (okadaic acid, microcystin, nodularin, calyculin A, and cantharidic acid), as well as inhibitor-2. The mutant enzyme exhibited a gain of function in the form of a 10-fold increased sensitivity toward okadaic acid that suggests this region is involved in toxin binding. Significant changes in sensitivity to inhibitor-2 and several of the other toxins were also observed, indicating that these may have a common binding region.
构建了一种嵌合突变体，其中兔肌肉蛋白磷酸酶-1的一个4氨基酸区域（GEFD，第274 - 277位氨基酸残基）被对应于兔蛋白磷酸酶-2A第267 - 270位氨基酸残基的YRCG序列所取代。这是基于对冈田酸抗性细胞中基因突变的研究结果，该突变导致蛋白磷酸酶-2A中的半胱氨酸向甘氨酸转化。蛋白磷酸酶-1的YRCG突变体被表达并纯化。从该突变体酶对几种毒素抑制剂（冈田酸、微囊藻毒素、节球藻毒素、花萼海绵诱癌素A和斑蝥素）以及抑制剂-2的敏感性方面对其性质进行了研究。该突变体酶表现出功能增强，对冈田酸的敏感性提高了10倍，这表明该区域参与毒素结合。还观察到对抑制剂-2和其他几种毒素的敏感性有显著变化，表明它们可能有一个共同的结合区域。
