IMPROVED METHOD FOR PREPARATION OF UBIQUITIN-LIGATED LYSOZYME AS SUBSTRATE OF ATP-DEPENDENT PROTEOLYSIS

被引：29

作者：

TAMURA, T ^{[1
]}

TANAKA, K ^{[1
]}

TANAHASHI, N ^{[1
]}

ICHIHARA, A ^{[1
]}

机构：

[1] UNIV TOKUSHIMA,INST ENZYME RES,TOKUSHIMA 770,JAPAN

来源：

FEBS LETTERS | 1991年 / 292卷 / 1-2期

关键词：

ATP; UBIQUITIN; LYSOZYME; UBIQUITIN PROTEIN LIGASE; ATP-DEPENDENT; 26; S-PROTEASE;

D O I：

10.1016/0014-5793(91)80856-X

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat-denatured I-125-labeled lysozyme was highly ubiquitinated by incubation at pH 9.0 with a ubiquitin-protein ligase system consisting of E1, E2 and E3 that had been partially purified from rabbit reticulocytes by affinity chromatography with ubiquitin as a ligand. The resulting conjugates were separated from free lysozyme and other proteins by successive chromatographies on anion and cation ion-exchange resins. The ubiquitinated I-125-lysozymes recovered in the fraction not adsorbed to either resin served as an efficient substrate for ATP-dependent proteolysis in a reticulocyte lysate or with a purified 26 S protease complex. By the present method, I-125-lysozyme-Ub conjugates can be prepared in 3 h with a high yield of 15-20%.

引用

页码：154 / 158

页数：5

共 12 条

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