Effect of glucose starvation on the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of yeast.

Yeast cells growing on mineral medium plus ammonia and glucose contained high levels of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase activity, as measured in crude extracts. After suspension of cells in fresh medium lacking glucose, there was a loss of the glutamate dehydrogenase activity. Loss of activity was inhibited by 2,4-dinitrophenol, sodium azide, iodoacetic acid, and cycloheximide. The enzyme activity was restored when glucose was added back to the medium, and this recovery was fully prevented in the presence of cycloheximide.     

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

Inactivation of the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase from Saccharomyces cerevisiae during carbon starvation occurs with a simultaneous loss of enzyme protein and enzyme activity.     

The culture of plant cells with ammonium salts as the sole nitrogen source

Soybean cell suspension cultures grew on defined media with ammonium as the sole nitrogen source if Krebs cycle acids were added. Satisfactory growth was obtained with ammonium salts of citrate, malate, fumarate, or succinate, when compared with the regular medium containing nitrate and ammonium. Little or no growth occurred when ammonium salts of shikimate, tartrate, acetate, carbonate, or sulfate were used. The cells also grew well with l-glutamine as nitrogen source. The specific activities of glutamine synthetase and isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate) were lower than in cells grown on a nitrate medium, but ammonium enhanced the activity of glutamate dehydrogenase. Cells of soybean, wheat, and flax have been cultured for an extended period on the ammonium citrate medium.     

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.     

Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.     

Decrease in nitrate reductase activity in extracts of Trichoderma viride Incubated with chlorides.

Reduced nicotinamide adenine dinucleotide phosphate-dependent nitrate reductase activity in crude extracts of Trichoderma virde was significantly inhibited by physiological concentrations of ammonium chloride, sodium chloride, and potassium chloride, but not by ammonium or sodium sulfate. The chloride inhibition of nitrate reductase activity increased in a linear manner with chloride concentration.     

Regulation of the nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenases of Saccharomyces cerevisiae

Saccharomyces cerevisiae contains two distinct l-glutamate dehydrogenases. These enzymes are affected in a reciprocal fashion by growth on ammonia or dicarboxylic amino acids as the nitrogen source. The specific activity of the nicotinamide adenine dinucleotide phosphate (NADP) (anabolic) enzyme is highest in ammonia-grown cells and is reduced in cells grown on glutamate or aspartate. Conversely, the specific activity of the nicotinamide adenine dinucleotide (NAD) (catabolic) glutamate dehydrogenase is highest in cells grown on glutamate or aspartate and is much lower in cells grown on ammonia. The specific activity of both enzymes is very low in nitrogen-starved yeast. Addition of the ammonia analogue methylamine to the growth medium reduces the specific activity of the NAD-dependent enzyme and increases the specific activity of the NADP-dependent enzyme.     

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.     

Effects of ammonium, L-glutamate, and L-glutamine on nitrogen catabolism in Aspergillus nidulans

During growth of Aspergillus nidulans in medium containing ammonium the specific activities of most enzymes involved in catabolism of nitrogen sources are low (ammonium repression). The gdhA10 lesion, which results in loss of nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase activity, has been shown to lead to partial relief of ammonium repression of three amidase enzymes as well as histidase. The areA102 lesion led to altered levels of these enzymes but did not greatly affect ammonium repression. The double mutant areA102,gdhA10 was almost completely insensitive to ammonium repression of two of the amidase enzymes and histidase. This suggests that an interaction between the areA and gdhA genes in determining responses to ammonium occurs. Growth of mycelium in medium containing l-glutamate has been found to result in lowered levels of all four enzymes, and this occurs in strains insensitive to ammonium repression. Very strong repression in all strains occurred during growth in medium containing l-glutamine. Relief of these repressive effects of glutamate and glutamine was blocked by cycloheximide. Glutamate and glutamine had similar effects on the production of extracellular protease activity, and growth on glutamine led to low levels of urate oxidase. In contrast to the above enzymes, nitrate reductase was insensitive to the effects of glutamine and glutamate, even though this enzyme is very sensitive to ammonium repression. Although other possibilities exist, it is suggested that there may be mechanisms of general control of nitrogen-catabolic enzymes other than ammonium repression.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Metabolism of the anaerobic formation of succinic acid bySaccharomyces cerevisiae

1. Succinic acid is formed in amounts of 0.2–1.7 g/l by fermenting yeasts of the genusSaccharomyces during the exponential growth phase. No differences were observed between the various species, respiratory deficient mutants and wild type strains.
2. At low glucose concentrations the formation of succinic acid depended on the amount of sugar fermented. However, the nitrogen source was found to be of greater importance than the carbon source.
3. Of all nitrogen sources, glutamate yielded the highest amounts of succinic acid. Glutamate led to an oxidative and aspartate to a reductive formation of succinic acid.
4. A reductive formation of succinic acid by the citric acid cycle enzymes was observed with malate. This was partially inhibited by malonate. No evidence was obtained that the glyoxylate cycle is involved in succinic acid formation by yeasts.
5. Anaerobically grown cells ofSaccharomyces cerevisiae contained α-ketoglutarate dehydrogenase. Its activity was found in the 175000 x g sediment after fractionated centrifugation. The specific activity increased 6-fold after growth on glutamate as compared with cells grown on ammonium sulfate.
6. The specific activities of malate dehydrogenase, fumarase, succinate dehydrogenase, succinylcoenzymeA synthetase, α-ketoglutarate dehydrogenase and glutamate dehydrogenase (nicotinamide adenine dinucleotide dependent) were determined in yeast cells grown on glutamate or ammonium sulfate. Similar results were obtained with a wild type strain and a respiratory deficient mutant. The latter did not contain succinate dehydrogenase.
7. In fermenting yeasts succinic acid is mainly formed from glutamate by oxidation.

     

Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa.

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.     

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.     

Regulation of Bacteriochlorophyll Synthesis by Oxygen in Respiratory Mutants of Rhodopseudomonas capsulata

Respiratory mutants of the facultative photosynthetic bacterium Rhodopseudomonas capsulata were used to investigate the mechanism of (reversible) inhibition of bacteriochlorophyll (BChl) synthesis by molecular oxygen. Although mutant strain M5 lacks cytochrome oxidase activity, it closely resembles the parental wild-type strain in respect to the effect of O(2) on BChl formation. This observation does not support an earlier hypothesis that O(2) regulates BChl synthesis through an effect on the redox state of a component of the respiratory electron transport system. Mutant strain M2 shows normal cytochrome oxidase activity, but lacks both reduced nicotinamide adenine dinucleotide and succinate dehydrogenase activities; relative to the parental strain, BChl synthesis in M2 is more sensitive to O(2) inhibition. The foregoing and results of related experiments can be accounted for by a revised interpretation of the O(2) effect, which proposes that O(2) directly inactivates a "factor" necessary for BChl formation and that, at relatively low O(2) tension, the inactivation can be reversed by a flow of electrons (derived from reduced nicotinamide adenine dinucleotide and succinate) diverted from a portion of the electron transport system delimited by the mutational blocks in M2 and M5.     

Multiple sites of action of cycloheximide in addition to inhibition of protein synthesis in Physarum polycephalum

The specificity of action of cycloheximide was tested using a cycloheximide resistant mutant of Physarum polycephalum. This resistance has previously been shown to reside with the ribosomes, making cytoplasmic protein synthesis refractile to the action of the drug. We show here that cycloheximide in the mutant strain causes specific alterations in metabolism without influencing the growth rate. These are: 1. lowered specific activity of glutamate dehydrogenase during starvation, 2. alteration of the molecular weight of glutamate dehydrogenase, 3. inhibition of uptake of amino acids from the medium into the internal pools. Possible explanations for these effects of cycloheximide outside of protein synthesis per se are considered. We conclude that cycloheximide may not be considered a specific inhibitor of protein synthesis, and that a causal relationship between protein synthesis and any biological process cannot be claimed unless such specificity is demonstrated in each case, preferably by use of mutants.     

Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent-l-glutamate dehydrogenase [EC 1.4.1.4; l-glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l-glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis, and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l-glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l-glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus, irrespective of nutritional conditions or of the physiological age of cells.     

Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.     

Induction and Repression of Nitrate Reductase in Neurospora crassa

Synthesis of wild-type Neurospora crassa assimilatory nitrate reductase is induced in the presence of nitrate ions and repressed in the presence of ammonium ions. Effects of several Neurospora mutations on the regulation of this enzyme are shown: (i) the mutants, nit-1 and nit-3, involving separate lesions, lack reduced nicotinamide adenine dinucleotide (NADPH)-nitrate reductase activity and at least one of three other activities associated with the wild-type enzyme. The two mutants do not require the presence of nitrate for induction of their aberrant nitrate reductases and are constitutive for their component nitrate reductase activities in the absence of ammonium ions. (ii) An analog of the wild-type enzyme (similar to the nit-1 enzyme) is formed when wild type is grown in a medium in which molybdenum has been replaced by vanadium or tungsten; the resulting enzyme lacks NADPH-nitrate reductase activity. Unlike nit-1, wild type produced this analog only in the presence of nitrate. Contaminating nitrate does not appear to be responsible for the observed mutants' activities. Nitrate reductase is proposed to be autoregulated. (iii) Mutants (am) lacking NADPH-dependent glutamate dehydrogenase activity partially escape ammonium repression of nitrate reductase. The presence of nitrate is required for the enzyme's induction. (iv) A double mutant, nit-1 am-2, proved to be an ideal test system to study the repressive effects of nitrogen-containing metabolites on the induction of nitrate reductase activity. The double mutant does not require nitrate for induction of nitrate reductase, and synthesis of the enzyme is not repressed by the presence of high concentrations of ammonium ions. It is, however, repressed by the presence of any one of six amino acids. Nitrogen metabolites (other than ammonium) appear to be responsible for the mediation of "ammonium repression."     

Mutants of Aspergillus nidulans lacking nicotinamide adenine dinucleotide-specific glutamate dehydrogenase.

Ten mutants of Aspergillus nidulans lacking nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GDH) have been isolated, and their mutations (gdhB1 through gdhB10) have been shown to lie in the gdhB gene. In addition, a temperature-sensitive gdhB mutant (gdhB11) has been isolated. A revertant (designated R-5) of the mutant gdhB1 bears an additional lesion in the gdhB gene and has altered NAD-GDH activity with altered Km values for ammonia or ammonium ions and for alpha-ketoglutarate. These results suggest that gdhB specifies a structural component for NAD-GDH. The growth characteristics of gdhB mutants indicate the routes by which amino acids are utilized as nitrogen and carbon energy sources. The properties are described of the double mutants bearing the mutations gdhB1 and gdhA1 or tamA119, which have low NADP-GDH activity.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.