DIMER-DIMER BINDING REGION IN BETA-GALACTOSIDASE
DIMER-DIMER BINDING REGION IN BETA-GALACTOSIDASE
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DOI：
10.1021/bi00570a002
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发表时间：
1979-01-01
期刊：
影响因子：
2.9
通讯作者：
ZABIN, I
中科院分区：
文献类型：
--
作者：
CELADA, F;ZABIN, I
α-β中的互补半乳糖苷酶[EC 3.2.1.23，大肠杆菌的]是通过加入α-半乳糖苷酶来恢复酶活性。供体CNBr 2，从多肽的氨基酸残基3-92，与来自lacZ缺失突变菌株M15的失活M15蛋白质结合。M15蛋白缺少残基11-41，并且是二聚体;活性复合物，像天然的β-半乳糖苷酶是四聚体。β-二聚体结合区通过α-半乳糖苷酶的蛋白水解和免疫学研究鉴定了半乳糖苷酶。互补用胰蛋白酶进行蛋白水解实验。治疗天然β-半乳糖苷酶与胰蛋白酶反应，然后混合物与CNBr反应，产生完整的CNBr 2，如通过其补充M15蛋白的能力所测量的。当尿素变性β-CNBr 2时，不能获得活性CNBr 2。半乳糖苷酶以相同的方式处理。因此，对应于CNBr 2的片段显然被埋在折叠的蛋白质中。用抗CNBr 2、胰蛋白酶肽T8（残基60-140）和CNBr 3（残基93-187）的抗体进行免疫学实验。抗CNBr 2和抗T8结合M15蛋白，但不结合β-半乳糖苷酶，表明该区域暴露在二聚体中。抗-CNBr 2而非抗-T8或抗-CNBr 3抑制α-CNBr的形成。补充酶。序列的早期部分，在对应于CNBr 2的区段内，显然参与二聚体-二聚体相互作用。
.alpha.-Complementation in .beta.-galactosidase [EC 3.2.1.23, of Escherichia coli] is the restoration of enzyme activity by adding .alpha. donor CNBr2, from amino acid residues 3-92 of the polypeptide, to inactive M15 protein from the lacZ deletion mutant strain M15. M15 protein lacks residues 11-41 and is a dimer; the active complex, like native .beta.-galactosidase, is tetrameric. A dimer-dimer binding region in .beta.-galactosidase was identified by proteolytic and immunologic studies of .alpha.-complementation. Proteolytic experiments were carried out with trypsin. Treatment of native .beta.-galactosidase with trypsin, followed by reaction of the mixture with CNBr, yields intact CNBr2 as measured by its ability to complement M15 protein. Active CNBr2 is not obtained when urea-denatured .beta.-galactosidase is treated in the same way. Therefore, the segment corresponding to CNBr2 is apparently buried within the folded protein. Immunologic experiments were carried out with antibodies against CNBr2, tryptic peptide T8 (residues 60-140) and CNBr3 (residues 93-187). Anti-CNBr2 and anti-T8 bind to M15 protein but not to .beta.-galactosidase, indicating that this area is exposed in the dimer. Anti-CNBr2, but not anti-T8 or anti-CNBr3, inhibits the formation of .alpha.-complemented enzyme. An early part of the sequence, within the segment corresponding to CNBr2, is apparently involved in dimer-dimer interaction.