PROTEOLYSIS REGULATION IN SPORULATING BACILLUS SUBTILIS CELLS

这项研究的长期目标是用生物化学术语描述 蛋白质降解的过程，发生在相对较高的速率（k1 = 0.15- 0.22/h）。 作为蛋白 退化似乎是所有类型的基本和普遍的过程， 对于细胞，它的详细描述在生物学中具有基本的重要性。 的 细菌枯草芽孢杆菌，建议在这里进行研究，具有特殊的 非常好地表征了中心代谢途径的优点， 遗传学 具体而言，（a）将采用分子遗传学方法 克隆并测序B。枯草钙调蛋白，目的是 确定该蛋白质是否在观察到的Ca 2+依赖性中起作用 蛋白质分解 潜在的医学重要性是我们的B。 美国国立卫生研究院的Jan Wolff最近发现，枯草钙调素 刺激百日咳杆菌腺苷酸环化酶。 (b)一种新的蛋白水解酶 基于使用细胞蛋白质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将被 测试它们中的任何一个是否对Ca 2+、嘌呤核苷酸或其他 潜在的能量信号，因为整个蛋白质水解过程已经 发现是能量和Ca 2+依赖的。 (c)我们将研究 几种蛋白酶抑制剂对蛋白质降解速率的影响。 (d)最后， 2 e将研究最近发现的依赖于钙离子的 转氨酶作为B中的“标记”反应。枯草杆菌细胞;在 特别是，我们将确定这样的活动是否可以提高利率， 体外酶的蛋白质水解。 氧气的可能作用 代谢物（超氧化物和过氧化物）也将在体外 和体内用于可能的标记活性。

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.