REGULATION OF PROTEOLYSIS IN BACILLUS SUBTILIS CELLS

研究的目的是了解细胞内的性质和控制 蛋白质降解，特别是在生物发育过程中。在.期间 芽胞发育枯草芽孢杆菌细胞经历蛋白质速率 一级速率常数高达0.23h-1的降解 并依赖于钙离子的流入。我们建议研究如何控制 这种生物体的主要蛋白酶，一种依赖于钙离子的丝氨酸酶，它 在产孢期经历戏剧性的激活。首先，提出了 为了分离和制备该蛋白水解酶的抗体，测定其 酪蛋白的特异性切割位点和筛选出一系列 目的是找到一种高度特异的抑制剂，这将是 用来评估蛋白酶在蛋白质降解中的作用。第二个我们 建议分离和研究一种胞质蛋白抑制物 已知在产孢子过程中活性下降的蛋白水解酶。在……里面 与最近在枯草杆菌中发现的一种钙调蛋白类似，也 发现是一种抑制蛋白酶，将对其进行分离和研究。 分子量、等电点、氨基酸组成和 将这两种蛋白质的氨基末端残基进行比较以确定 如果这些蛋白质是相同的。将针对每个人制备抗血清 并用于通过火箭免疫电泳法或放射免疫法测定 蛋白质的出现和消失的数量和次数(S) 在生长和产孢期。抗血清也将被用来观察是否有 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。最后给出了一个方案 建议从枯草杆菌中克隆钙调蛋白基因，第一例 将革兰氏阳性原核表达载体中的钙调蛋白连接到其 氯霉素可诱导表达。其目的将是确定 细胞钙调蛋白水平升高的影响(由 氯霉素)对蛋白水解率的影响。一个可能的观点是 这项研究的医学意义在于在肌营养不良症方面的最新工作 提示钙离子的内流可能是导致血压升高的原因之一 蛋白水解性破坏肌原纤维。

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.