PURIFICATION OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

ATP/泛素依赖性蛋白酶的纯化

• 批准号: 3291832
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 14.98万
• 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/3291832
• 关键词: adenosine triphosphate; affinity chromatography; antibody; enzyme complex; enzyme structure; enzyme substrate; fibroblasts; fluorescence spectrometry; gel electrophoresis; ion exchange chromatography; ligase; lysozyme; peptidases; reticulocytes; tissue /cell culture

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.

本提案的长期目标是通过以下方式阐明机制： 这些细胞降解它们的细胞内蛋白质。 泛素，一个小的 在进化过程中序列非常保守的蛋白质， 参与动物细胞内ATP依赖性蛋白水解。 我们有 从氯化血红素抑制的兔中纯化的泛素-溶菌酶缀合物 网织红细胞裂解物，我们已经表明，这些共轭物迅速 在返回到未抑制的裂解物时降解。 更重要的是 偶联物是一种大的ATP依赖性蛋白酶的底物， 降解游离的溶菌酶分子。 使用传统的生物化学 利用分级分离技术，我们将ATP依赖性蛋白纯化至同质 蛋白酶，表征它并产生抗体。这些抗体 反过来，将是有用的试剂，用于发现是否依赖ATP的 蛋白酶参与大多数细胞蛋白质的降解，或者仅参与细胞蛋白质的降解。 子集 此外，我们将纯化足够量的泛素 激活酶产生抗体。 最后我们将 继续分析泛素-溶菌酶结合物的结构， 试图找出它们为什么如此巨大。