Studies on the regulation of aspartokinase II from Bacillus subtilis

Aspartokinase II from B. subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t$\sb{1/2}$ = 65 min), followed by amino acid starvation (t$\sb{1/2}$ = 80 min), and glucose limitation (t$\sb{1/2}$ = 120 min). Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy and protein synthesis, but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. Cross reaction of anti-aspartokinase II antibodies with a protein similar in sequence to glyceraldehyde-3-phosphate from B. subtilis and E. coli was observed.In addition, a previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr = 120,000, which accounts for previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid inhibitable isozyme, was constant under all growth conditions examined.U of I OnlyETDs are only available to UIUC Users without author permissio