Evidence for the Degradation of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutamate Dehydrogenase of Candida utilis During Rapid Enzyme Inactivation

Abstract

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.

Proton production and consumption pathways in yeast metabolism. A chemostat culture analysis

“…The NADP-dependent GDH (EC 1.4.1.4), which catalyses the direct reaction towards the production of glutamate, and NADdependent GDH (EC 1.4.1.2), which catalyses the inverse reaction (Smith et al, 1975;Large, 1986). The activities of both enzymes are regulated by glucose and different nitrogen sources (Hemmings, 1978;Bogonez et al, 1985;Large, 1986). Thus, in the presence of ammonia or nitrogen sources which result in intracellular ammonia generation (i.e.…”

“…In the presence of these nitrogen sources, GDH drives the reaction towards the incorporation of nitrogen into the cell in the form of glutamate. The fact that the enzyme is induced by glucose, being rapidly inactivated in the absence of the sugar (Hemmings, 1978;Mazon, 1978) can reflect the crucial role of the enzyme in the coordination between carbon and nitrogen metabolism. On the basis of its regulatory properties, glutamate dehydrogenase has been claimed to play a crucial role as an anabolic route (Polakis and Bartley, 1965).…”

Regulation of Saccharomyces cerevisiae nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase by proteolysis during carbon starvation

“…Taken together, these data suggest that the NADP-GDH from S. cerevisiae is selectively proteolyzed during starvation for glucose. Thus, inactivation of the NADP-GDH from S. cerevisiae and C. utilis is apparently regulated in a similar manner during carbon starvation (4,7). Transfer of cultures of yeast to a medium deficient in a readily utilizable carbon source results in the activation of a specific NADP-GDH-degrading system.…”

“…There are several examples of the in vivo inactivation of microbial enzymes, in which it has been demonstrated that cross-reactive protein disappears from cells at the same rate as enzymatic activity: glycogen phosphorylase from Dictyostelium discoideum (13), phosphoribosylanthranilate isomerase-indoleglycerol phosphate from Escherichia coli (8), aspartate transcarbamylase from Bacillus subtilis (6), malate dehydrogenase from S. cerevisiae (9), and NADP-GDH from C. utilis (4). In all these systems no evidence for the production of fragments derived from the native enzyme was found.…”

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

“…NADP-GDH catalyzes the main aminating pathway in yeast [1], but, despite this key position, the regulatory properties of the enzyme are poorly understood. Carbon starvation results in an inactivation of the enzyme from S. cerevisiae or Candida utilis [2][3][4][5][6] that is accompanied by a loss of crossreacting material against specific antibodies, indicating that the protein undergoes a degradation process [3][4][5][6].…”