REGULATION OF PROTEOLYSIS IN BACILLUS SUBTILIS CELLS

枯草芽孢杆菌细胞中蛋白水解的调节

• 批准号: 3134981
• 负责人: JAMES H HAGEMAN
• 金额: $ 8.28万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3134981
• 关键词: Bacillus subtilis; bacterial genetics; genetic manipulation; genetic recombination; immunoelectrophoresis; microorganism growth; microorganism metabolism; molecular cloning; molecular weight; monoclonal antibody; muscular dystrophy; peptidases; protease inhibitor; proteolysis; radioimmunoassay; spores

Research is aimed at understanding the nature and control of intracellular protein degradation, especially during biological development. During spore development Bacillus subtilis cells undergo rates of protein degradation for which first order rate constants are as high as 0.23 h-1 and are dependent on an influx of Ca2+. We propose to study the control of the major proteinase of this organism, a Ca2+-dependent serine enzyme which undergoes a dramatic activation during sporulation. First, it is proposed to isolate and prepare antibodies against this protease, determine its sites of specificity for cleavage of casein and screen a series of inhibitors with the object of finding a highly specific one, which would be used to evaluate the protease's role in protein degradation. Second we propose to isolate and study a cytoplasmic protein inhibitor of the protease which is known to decline in activity during sporulation. In parallel a calmodulin protein, recently discovered in B. subtilis and also found to be an inhibitor of the protease, will be isolated and studies. The molecular weights, isoelectric points, amino acid compositions and amino terminal residues of these two proteins will be compared to determine if these proteins are identical. Antisera will be prepared against each and used to determine, by rocket immuno-electrophoresis or radioimmunoassay the amount and times of appearance and disappearance of the protein(s) during growth and sporulation. Antisera will also be used to see if either or both of these proteins can be coprecipitated with the protease from cell extracts prepared at different stages of sporulation. Finally a scheme is proposed to clone the calmodulin gene from B. subtilis, the first example of a calmodulin in a Gram positive procryote, onto a plasmid whose expression can be induced by chloramphenicol. The aim will be to determine the effect of elevated cellular calmodulin levels (induced by chloramphenicol) on the rates of proteolysis. One possible point of medical relevance of this study is that in muscular dystrophy recent work has suggested that the influx of Ca2+ is a possible cause of the elevated proteolytic destruction of the myofibrils.

研究旨在了解细胞内的性质和控制 蛋白质降解，尤其是在生物发育过程中。 期间 孢子发育 枯草芽孢杆菌细胞经历蛋白质代谢率 一阶速率常数高达 0.23 h-1 的降解 并且依赖于 Ca2+ 的流入。 我们建议研究控制 该生物体的主要蛋白酶，一种 Ca2+ 依赖性丝氨酸酶， 在孢子形成过程中经历戏剧性的激活。 首先，建议 分离并制备针对该蛋白酶的抗体，确定其 酪蛋白裂解的特异性位点并筛选一系列 抑制剂的目的是找到一种高度特异性的抑制剂，这将是 用于评估蛋白酶在蛋白质降解中的作用。 第二个我们 提议分离并研究一种细胞质蛋白抑制剂 已知在孢子形成过程中活性下降的蛋白酶。 在 与最近在枯草芽孢杆菌中发现的钙调蛋白类似 发现是蛋白酶的抑制剂，将被分离和研究。 分子量、等电点、氨基酸组成等 将比较这两种蛋白质的氨基末端残基以确定 如果这些蛋白质是相同的。 将针对每种情况准备抗血清 并用于通过火箭免疫电泳或放射免疫分析法进行测定 蛋白质出现和消失的数量和次数 在生长和产孢期间。 抗血清也将用于查看是否 或者这两种蛋白质都可以与细胞中的蛋白酶共沉淀 在孢子形成的不同阶段制备的提取物。 最后一个方案是 提议从枯草芽孢杆菌中克隆钙调蛋白基因，第一个例子 将革兰氏阳性原细胞中的钙调蛋白转移到质粒上，其 表达可由氯霉素诱导。 目的是确定 细胞钙调蛋白水平升高的影响（由 氯霉素）对蛋白水解速率的影响。 一个可能的点是 这项研究的医学相关性在于，在肌营养不良症方面最近的工作 表明Ca2+的流入可能是导致升高的原因 肌原纤维的蛋白水解破坏。