PROTEOLYSIS REGULATION IN SPORULATING BACILLUS SUBTILIS CELLS

枯草芽孢杆菌芽孢细胞中的蛋白水解调节

• 批准号: 6271585
• 负责人: JAMES H HAGEMAN
• 金额: $ 4.35万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271585
• 关键词: Bacillus subtilis; aspartate carbamoyltransferase; bioenergetics; catalase; endopeptidases; enzyme substrate; high performance liquid chromatography; interferometry; microorganism metabolism; peptidases; peroxides; protease inhibitor; protein degradation; protein purification; sporogenesis

The long term goal of the research is to describe in biochemical terms the process of protein degradation which occurs at relatively high rates (k1 = 0.15 - 0.22/h) during formation of the bacterial spore. As protein degradation appears to be a fundamental and universal process in all types of cells, its detailed description is of basic importance in biology. The bacterium Bacillus subtilis, proposed for study here, has the particular advantages of very well characterized central metabolic pathways and genetics. Specifically, (a) molecular genetic approaches will be applied to clone and sequence the B. subtilis calmodulin with the objective of determining whether this protein has a role in the observed Ca2+ dependence of proteolysis. Of potential medical importance is the fact that our B. subtilis calmodulin was recently found by Jan Wolff at NIH to strongly stimulate Bordetella pertusis adenylate cyclase. (b) A new proteolytic assay based on using cellular proteins as the substrate will be used to screen cells for undetected proteinases; any proteinases detected will be tested to see if any of them respond to Ca2+, purine nucleotides or other potential energy signals, since the overall proteolysis process has been found to be energy and Ca2+ dependent. (c) We will examine the effects of several protease inhibitors on rates of protein degradation. (d) Finally, 2e will examine the possible role of the recently discovered Ca2+ dependent transglutaminase as a "marking" reaction in B. subtilis cells; in particular we will determine whether such an activity can enhance the rate of proteolysis of enzymes in vitro. The possible role of oxygen metabolites (superoxide and peroxide) will also be examined both in vitro and in vivo for possible marking activity.

该研究的长期目标是用生化术语描述 以相对较高的速率发生的蛋白质降解过程 (k1 = 0.15 - 0.22/h）在细菌孢子形成期间。 作为蛋白质 退化似乎是所有类型的基本且普遍的过程 对于细胞来说，其详细描述在生物学中具有基本重要性。 这 枯草芽孢杆菌（Bacillus subtilis）被提议在这里进行研究，它具有特殊的 特征明确的中心代谢途径的优点和 遗传学。 具体来说，（a）将应用分子遗传学方法 对枯草芽孢杆菌钙调蛋白进行克隆和测序，目的是 确定该蛋白质是否在观察到的 Ca2+ 依赖性中起作用 的蛋白水解作用。 具有潜在医学重要性的是我们的 B. 美国国立卫生研究院 (NIH) 的简·沃尔夫 (Jan Wolff) 最近发现枯草芽孢杆菌钙调蛋白能够强烈 刺激百日咳博德特氏菌腺苷酸环化酶。 (b) 一种新的蛋白水解酶 基于使用细胞蛋白作为底物的测定将用于 筛选细胞中未检测到的蛋白酶；任何检测到的蛋白酶都会被 测试它们是否对 Ca2+、嘌呤核苷酸或其他物质有反应 势能信号，因为整个蛋白水解过程已经 发现与能量和 Ca2+ 相关。 (c) 我们将研究以下措施的影响： 几种蛋白酶抑制剂对蛋白质降解率的影响。 (d) 最后， 2e 将检查最近发现的 Ca2+ 依赖性的可能作用 转谷氨酰胺酶作为枯草芽孢杆菌细胞中的“标记”反应；在 特别是我们会确定这样的活动是否可以提高速率 体外酶的蛋白水解作用。 氧气的可能作用 代谢物（超氧化物和过氧化物）也将在体外进行检查 以及体内可能的标记活性。