REGULATION OF PROTEOLYSIS IN BACILLUS SUBTILIS CELLS

点击翻译按钮获取中文摘要

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.