PURIFICATION OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

ATP/泛素依赖性蛋白酶的纯化

• 批准号: 3291836
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 16.81万
• 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/3291836
• 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.

这一提议的长期目标是通过以下方式阐明机制(S) 这些细胞会降解它们细胞内的蛋白质。泛素，一种小的 其序列在进化过程中非常保守的蛋白质， 与动物细胞内依赖三磷酸腺苷的蛋白质分解有关。我们有 氯化高铁血红素抑制兔的纯化泛素-溶菌酶结合物 网织红细胞裂解物，我们已经证明这些结合物很快 在返回到不受限制的裂解物后降解。更重要的是， 结合物是一种大的、依赖于ATP的蛋白酶的底物，这种酶不 降解游离溶菌酶分子。使用传统的生物化学 分离技术，我们将提纯到均一的依赖于ATP的 蛋白水解酶，对其进行表征并产生抗体。这些抗体 反过来，将成为有用的试剂来发现依赖于ATP的 蛋白水解酶参与了大多数细胞蛋白质的降解，或者只有一个 子集。此外，我们还将提纯足够数量的泛素 激活酶以产生抗体。最后，我们会 继续分析泛素-溶菌酶结合物的结构 试着找出它们为什么会异常大。