大肠杆菌β-半乳糖苷酶的热片段化。α互补和两种非互补多肽组分的分离与表征。

Thermal fragmentation of Escherichia coli beta-galactosidase. Isolation and characterization of an alpha-complementing and two non-complementing polypeptide fractions.

作者信息

Marinkovic D V, Marinkovic J N, Tang J

出版信息

Int J Pept Protein Res. 1976;8(6):533-42.

Abstract

Carboxymethylated Escherichia coli beta-galactosidase EC 3.2.1.23 could be broken to polypeptides of fairly uniform size (average molecular weight about 22,000 daltons) by heating for less than or equal to 8 h at 100 degrees C and pH 7.5 IN 8 M-urea. Using phosphocellulose chromatography in NaCl-urea gradients, the resulting polypeptide mixture could be resolved in three fractions essentially homogeneous by disc gel electrophoresis in urea at several pH values, and by isoelectric focusing. One of these fractions was active as alpha-donor in in vitro complementation of beta-galactosidase activity with Escherichia coli mutant M15; this activity was largely retained after CNBr cleavage. All three fractions carried arginine as carboxyl-terminal amino acid. No significant amount of any specific amino could be detected in NH2-terminal position.

摘要

羧甲基化的大肠杆菌β-半乳糖苷酶（EC 3.2.1.23）在8M尿素中，于100℃、pH 7.5条件下加热8小时或更短时间，可断裂成大小相当均匀的多肽（平均分子量约22,000道尔顿）。利用磷酸纤维素在NaCl-尿素梯度中的层析法，所得多肽混合物可通过在几个pH值的尿素中进行圆盘凝胶电泳以及等电聚焦，分离为三个基本均一的组分。其中一个组分在与大肠杆菌突变体M15的β-半乳糖苷酶活性的体外互补中作为α供体具有活性；该活性在溴化氰裂解后大部分得以保留。所有三个组分的羧基末端氨基酸均为精氨酸。在氨基末端位置未检测到任何特定氨基酸的显著量。