PROTEOLYSIS REGULATION IN SPORULATING BACILLUS SUBTILIS CELLS

这项研究的长期目标是用生化术语描述 蛋白质以相对较高的速率降解的过程(k1= 0.15-0.22/h)。作为蛋白质 退化似乎是所有类型的基本和普遍的过程 在细胞中，它的详细描述在生物学中具有基本的重要性。这个 建议研究的枯草芽孢杆菌具有特殊的 特征明确的中枢代谢途径的优势和 遗传学。具体而言，(A)将采用分子遗传学方法 目的：克隆枯草杆菌钙调素基因并进行序列测定 确定该蛋白是否在观察到的钙依赖中起作用 蛋白水解酶。具有潜在医学意义的事实是我们的B。 美国国立卫生研究院的简·沃尔夫最近发现枯草杆菌钙调蛋白强烈 刺激百日咳杆菌腺苷环化酶。(B)一种新的蛋白质水解剂 以细胞蛋白为底物的检测将被用于 筛选细胞中未检测到的蛋白酶；检测到的任何蛋白酶都将 测试它们中的任何一个是否对钙离子、嘌呤核苷酸或其他 势能信号，因为整个蛋白质分解过程 发现是能量和钙依赖的。(三)我们会研究 几种蛋白酶抑制剂对蛋白质降解率的影响。(D)最后， 2E将研究最近发现的钙依赖的可能作用 转谷氨酰胺酶在枯草杆菌细胞中的“标记”反应； 具体地说，我们将确定这样的活动是否可以提高 酶在体外的蛋白质分解。氧的可能作用 代谢产物(超氧化物和过氧化氢)也将在体外进行检测。 并在体内进行可能的标记活性。

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.