Evidence for the degradation of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of Candida utilis during rapid enzyme inactivation.
The nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADP-GDH) from the food yeast Candida utilis was found to be rapidly inactivated when cultures were starved of a carbon source. The addition of glutamate or alanine to the starvation medium stimulated the rate of inactivation. Loss of enzyme activity was irreversible since the reappearance of enzyme activity, following the addition of glucose to carbon-starved cultures, was blocked by cycloheximide. A specific rabbit antibody was prepared against the NADP-GDH from C. utilis and used to quantitate the enzyme during inactivation promoted by carbon starvation. The amount of precipitable antigenic material paralleled the rapid decrease of enzyme activity observed after transition of cells from NH(4) (+)-glucose to glutamate medium. No additional small-molecular-weight protein was precipitated by the antibody as a result of the inactivation, suggesting that the enzyme is considerably altered during the primary steps of the inactivation process. Analysis by immunoprecipitation of the reappearance of enzyme activity after enzyme inactivation showed that increase of NADP-GDH activity was almost totally due to de novo synthesis, ruling out the possibility that enzyme activity modulation is achieved by reversible covalent modification. Enzyme degradation was also measured during steady-state growth and other changes in nitrogen and carbon status of the culture media. In all instances so far estimated, the enzyme was found to be very stable and not normally subject to high rates of degradation. Therefore, the possibility that inactivation was caused by a change in the ratio of synthesis to degradation can be excluded.
D000641:Ammonia / Q000378:metabolism; D000906:Antibodies; D000963:Antimetabolites / Q000494:pharmacology; D002175:Candida / Q000201:enzymology*; D003513:Cycloheximide / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000032:analysis / Q000378:metabolism*; D005779:Immunodiffusion; D009249:NADP
D000906:Antibodies; D000963:Antimetabolites; D009249:NADP; D000641:Ammonia; D003513:Cycloheximide; D005969:Glutamate Dehydrogenase; D005947:Glucose