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|Title:||Degradation of Aspartate Transcarbamylase in Growing and Starved Bacillus Subtilis Cells|
|Author(s):||Bond, Richard William|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Aspartate transcarbamylase (ATCase) is degraded upon starvation of Bacillus subtilis cells for carbon or nitrogen. The rate of ATCase degradation in exponentially growing B. subtilis cells was determined by measurement of enzyme activity after the addition of uridine to repress further enzyme synthesis and by specific immunoprecipitation of the enzyme from cells grown in the presence of (('3)H)leucine. ATCase was degraded with a half-life of about 1.5h in cells growing on a glucose-salts minimal medium with NH(,4)('+) ions as the sole source of nitrogen. Replacement of NH(,4)('+) in this medium with a combination of the amino acids aspartate, glutamate, isoleucine, proline, and threonine reduced the degradation rate to an undetectable level. Various other amino acids had smaller effects on the rate of degradation. The carbon source also influenced the degradation rate, but to a smaller extent than the nitrogen source. The effects of these nutritional variables on the rate of bulk protein turnover in growing cells were generally similar to their effects on degradation of ATCase.
Addition of protein synthesis inhibitors to B. subtilis cells grown in minimal medium under conditions in which ATCase was either stable or unstable blocked the rapid degradation of ATCase. This is in contrast with results from cells grown in a supplemented nutrient broth medium. Degradation of ATCase was also deficient in growing and starved relA and relC mutants, but these effects were not correlated with differences in the intracellular level of guanosine polyphosphates.
An assay for ATCase-degrading activity in vitro was developed to test the involvement of ribosomally associated factors in ATCase degradation. Cells were fractionated, and activity was found in both the crude membrane and ribosomal fractions but not in the soluble fraction. The activity in vitro was not correlated with ATCase degradation in vivo. Activity was present in fractions from cells in which ATCase was stable. Also, ATCase degradation in vitro was not affected by mutations in the relA or relC proteins.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.
|Date Available in IDEALS:||2014-12-15|