Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.
The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003486:Cyanides / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000276:immunology / Q000378:metabolism*; D005973:Glutamine / Q000494:pharmacology*; D006041:Goats / Q000276:immunology; D005779:Immunodiffusion; D007700:Kinetics; D008433:Mathematics; D011233:Precipitin Tests; D011817:Rabbits / Q000276:immunology; D013696:Temperature; D013997:Time Factors
D003486:Cyanides; D005973:Glutamine; D005974:Glutamate-Ammonia Ligase; D005947:Glucose