PURIFICATION OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

ATP/泛素依赖性蛋白酶的纯化

• 批准号: 3291837
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 17.32万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291837
• 关键词: adenosine triphosphate; affinity chromatography; antibody; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; fibroblasts; fluorescence spectrometry; gel electrophoresis; ion exchange chromatography; laboratory rabbit; ligase; lysozyme; peptidases; reticulocytes; tissue /cell culture; ubiquitin

The long term goal of this proposal is to elucidate the mechanism(s) by which cells degrade their intracellular proteins. Ubiquitin, a small protein whose sequence has been remarkably conserved during evolution, is implicated in ATP-dependent proteolysis within animal cells. We have purified ubiquitin-lysozyme conjugates from hemin-inhibited rabbit reticulocyte lysates, and we have shown that these conjugates are rapidly degraded upon return to uninhibited lysates. More important, the conjugates are substrates for a large, ATP-dependent protease that does not degrade free lysozyme molecules. Using traditional biochemical fractionation techniques, we will purify to homogeneity the ATP-dependent protease, characterize it and produce antibodies to it. These antibodies will, in turn, be useful reagents for discovering whether the ATP-dependent protease is involved in the degradation of most cellular proteins or only a subset. In addition, we will purify sufficient quantities of the ubiquitin activating enzymes to produce antibodies to them. Finally, we will continue to analyze the structure of ubiquitin-lysozyme conjugates in an attempt to discover why they are anomalously large.

该提案的长期目标是通过以下方式阐明机制： 哪些细胞降解其细胞内蛋白质。 泛素，一种小 其序列在进化过程中显着保守的蛋白质是 参与动物细胞内 ATP 依赖性蛋白水解作用。 我们有 从血红素抑制的兔中纯化泛素-溶菌酶缀合物 网织红细胞裂解物，我们已经证明这些结合物可以快速 返回未抑制的裂解物后降解。 更重要的是， 缀合物是大型 ATP 依赖性蛋白酶的底物，该蛋白酶不 降解游离溶菌酶分子。 采用传统生化法 分馏技术，我们将纯化至同质的 ATP 依赖性 蛋白酶，对其进行表征并产生针对它的抗体。 这些抗体 反过来，将成为有用的试剂，用于发现 ATP 依赖性 蛋白酶参与大多数或仅一部分细胞蛋白质的降解 子集。 此外，我们将纯化足够量的泛素 激活酶以产生针对它们的抗体。 最后，我们将 继续分析泛素-溶菌酶缀合物的结构 试图找出它们异常大的原因。