Oxidative Inactivation of Bacillus Subtilis Aspartate Transcarbamylase in Vitro

214 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.Aspartate transcarbamylase (ATCase) is stable in growing Bacillus subtilis cells, but it is rapidly and selectively inactivated in stationary cells. It has been demonstrated that the inactivation requires the continuous generation of metabolic energy, but not nucleic acid or protein synthesis. Immunochemical studies indicated that ATCase is degraded simultaneously with inactivation in vivo.I have found three NADH-dependent cell-free systems which will inactivate ATCase. The first was B. subtilis membrane vesicles plus NADH, azide, and oxygen. Azide probably served primarily to inhibit the action of a membrane-associated catalase, implicating the involvement of H(,2)O(,2). However, ATCase was stable in the presence of H(,2)O(,2). Amino acid analysis of the reisolated inactivated ATCase revealed the loss of 1 to 2 histidine residues per subunit. The inactivated ATCase also possessed a new chromophore which absorbed in the ultraviolet. Inactivation of ATCase and chromophore formation were probably mediated by two independent vesicle-mediated phenomena, because inactivation required the presence of oxygen, but chromophore formation did not.The second cell-free system consisted of NADH, oxygen, and a flavoprotein, which was purified from stationary B. subtilis cells. Purification was accomplished by locating an activity which would inactivate ATCase in the presence of NADH and oxygen. The purified flavoprotein was shown to generate H(,2)O(,2) from NADH and oxygen.The third system consisted solely of NADH and H(,2)O(,2). Amino acid analysis the reisolated ATCase again revealed the loss of 1 to 2 histidine residues per subunit but the formation of chromophore did not occur. The participation of an activated oxygen species, possibly singlet oxygen, in the inactivation was indicated. It is probable that the inactivation seen in the other two systems actually is caused by this third system. ATCase that had been inactivated by the third system was not more susceptible than native ATCase to proteolysis by trypsin, chymotrypsin, or subtilisin. ATCase treated anaerobically with vesicles plus NADH (that is, active ATCase bearing the chromophore) was more susceptible than native to trypsin treatment only.The mechanisms and the possible relationship of these inactivation phenomena to the disappearance of ATCase in vivo are discussed.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD