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.
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