Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells.
Mixed-function oxidation of Escherichia coli glutamine synthetase has previously been suggested to mark the enzyme for intracellular degradation, and in vitro studies have demonstrated that oxidation renders the enzyme susceptible to proteolytic attack. In this study, the susceptibility of glutamine synthetase to degradation by purified proteases has been compared with the rate of degradation after microinjection into hepatoma cells. Upon exposure to an ascorbate mixed-function oxidation system the enzyme rapidly loses most of its activity, but further oxidation is required to cause susceptibility to extensive proteolytic attack either by a high-molecular-weight liver cysteine proteinase or by trypsin. The rate of degradation of biosynthetically 14C-labeled native and oxidized glutamine synthetase preparations after injection into hepatoma cells parallels their susceptibility to proteolysis in vitro. Native enzyme preparations and enzyme oxidatively inactivated, but not susceptible to extensive degradation by purified proteases, had similar intracellular half-lives; however, oxidized enzyme preparations that were susceptible to proteolytic breakdown in vitro were degraded almost ten times faster than the native enzyme within the growing hepatoma cells. These results suggest that the same features of the oxidized enzyme that render it susceptible to proteolysis in vitro are also recognized by the intracellular degradation system. In addition, they show that loss of enzyme activity does not necessarily imply decreased metabolic stability.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008845:Microinjections; D006899:Mixed Function Oxygenases / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000201:enzymology*
D006899:Mixed Function Oxygenases; D005974:Glutamate-Ammonia Ligase; D001205:Ascorbic Acid