Regulation of glutamate dehydrogenases during morphogenesis of Schizophyllum commune

The specific activities of two glutamate dehydrogenases (GDH), one requiring nicotinamide adenine dinucleotide (NAD) and the other specific for nicotinamide adenine dinucleotide phosphate (NADP), varied during growth of Schizophyllum commune as a function of the stage of the life cycle and the exogenous nitrogen source. During basidiospore germination on either glucose-NH(3) or glucose-glutamate medium, NADP-GDH increased six- to eightfold in specific activity, whereas NAD-GDH was depressed. During dikaryotic mycelial growth on either nitrogen source, the two GDH increased in a 1:1 ratio, whereas, during homokaryotic mycelial growth on glucose-NH(3), NADP-GDH activity was depressed and NAD-GDH increased six- to eightfold. Homokaryotic mycelium cultured on glucose-glutamate medium yielded high NADP-GDH activities and normal NAD-GDH activities. Intracellular NH(3) concentration and NADP-GDH activities were inversely related during spore germination and homokaryotic mycelium growth, whereas guanosine-5'-triphosphate (GTP) and l-glutamine specifically inhibited NAD- and NADP-GDH respectively in vitro. GTP inhibition was shown in extracts from cells at all stages of the life cycle. Basidiospore germling extracts contained an NADP-GDH essentially resistant to l-glutamine inhibition.     

Regulation of nitrogen-metabolizing enzymes in the commercial Mushroom Agaricus bisporus

Mycelium of Agaricus bisporus strain Horst U1 was grown in batch cultures on different concentrations of ammonium, glutamate, and glucose to test the effect of these substrates on the activities of NADP-dependent glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4), NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2.), and glutamine synthetase (GS, EC 6.3.1.2.). When grown on ammonium, the activities of NADP-GDH and GS were repressed. NAD-GDH activity was about 10 times higher than the activities of NADP-GDH and GS. At concentrations below 8 mM ammonium, NADP-GDH and GS were slightly derepressed. When glutamate was used as the nitrogen source, activities of NADP-GDH and GS were derepressed; compared with growth on ammonium, the activities of these two enzymes were about 10 times higher. Activities of GDHs showed no variation at different glutamate concentrations. Activity of GS was slightly derepressed at low glutamate concentrations. Growth of A. bisporus on both ammonium and glutamate as nitrogen sources resulted in enzyme activities comparable to growth on ammonium alone. Activities of NADP-GDH, NAD-GDH, and GS were not influenced by the concentration of glucose in the medium. In mycelium starved for nitrogen, the activities of NADP-GDH, NAD-GDH, and GS were derepressed, while in carbon-starved mycelium the activity of GS and both GDHs was repressed.     

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.     

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.     

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.     

Observations on the Regulation of Glutamate Dehydrogenase Activity in Coprinus lagopus

Extracts of the mycelium of Coprinus lagopus (sensu Buller)contain two glutamate dehydro-genases with different optimumpH values. One is assayed with nicotinamide adenine dinucleotide(NAD-GDH) and the other with nicotinamide adenine dinucleotidephosphate (NADP-GDH). Changes in specific activity of the enzymeswere investigated during the growth of both a monokaryon (H9)and a dikaryon (H9 x TC) in different media and after the transferof mycelium from one growth medium into another. In the lattercase the magnitude of the changes in enzyme activity could bealtered by modification of either the carbon or the nitrogensource in the transfer medium. It is concluded from the resultsobtained that neither glutamate nor the ammonium ion seems toregulate directly the synthesis of either enzyme. However, someof the results are in accordance with the view that a productof glucose metabolism represses the synthesis of the NAD-GDHand derepresses or induces that of the NADP-CDH and evidencethat this regulator is 2-oxoglutarate was obtained. It is alsoconcluded that the complete system of regulation must involvemore than one molecule.     

Importance of glutamate synthase in glutamate synthesis by soybean cell suspension cultures

The specific activities of glutamate synthase|EC 2.6.1.53, l-glutamine: alpha-ketoglutarate amino transferase (NADPH-oxidising)| and glutamine synthetase|EC 6.3.1.2, l-glutamate: ammonia ligase (ADP-forming)| extracted from soybean (Glycine max L.) cells grown in modified B5 medium were found to vary significantly in response to variations in the nitrogen content of the medium. The changes seen in specific activity levels could be correlated with similar patterns seen in the growth of the cells, in response to changes in the nitrogen content of the medium. By contrast, the specific activity of glutamate dehydrogenase|EC 1.4.1.2, l-glutamate: NAD(+) oxidoreductase (deaminating)|, was relatively low and invariant. Glutamate synthase was extracted from cells grown under optimal conditions, partially purified, and shown to have many properties in common with preparations of this enzyme extracted from other plant sources. Glutamate synthase was purified to homogeneity, using affinity chromatography on blue Sepharose.     

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.     

Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana.

The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

小球藻NADP-谷氨酸脱氢酶的cDNA克隆及转基因烟草分析

用RT-PCR方法从小球藻(Chlorella sorokiniana)中克隆了铵诱导表达的以辅酶Ⅱ为辅基的谷氨酸脱氢酶(NADP-GDH)基因的cDNA片段,DNA测序分析表明与已报道的该基因c DNA序列同源性为94%.将NADP-GDH基因先插入到SPDK621质粒的2CaMV35S启动子和Ω增强序列之后,然后将2CaMV35S-Ω-GDH-NOS表达单元构建到RokⅡ质粒的HindⅢ与Eco RⅠ之间,从而获得高效植物表达载体.将RokⅡ-GDH质粒转移到根癌土壤杆菌(Agro bacterium tumefaciens (Smith et Townsend) Conn) EHA105中,对烟草(Nico tiana tabacum L.)进行转化并得到阳性转化后代.对转基因烟草分析表明,在低氮培养基或在低氮蛭石中其生长速度和叶片数明显高于对照;铵毒性实验表明,无论在低铵或高铵条件下,接种在MS固化培养基上的转基因绿叶圆片存活时间长,叶绿素含量高.这些结果说明外源NADP-GDH增强了植物对氮素的吸收和利用.另外,转化后代还表现了对除草剂膦化麦黄酮(PPT)具有较强的抗性;培养在含有不同浓度PPT的MS固化培养基上的转基因绿叶圆片,其愈伤化程度明显高于对照;在MS培养基中用0.5 μg/mL 的PPT可以代替卡那霉素对转化后代进行筛选,这暗示 NADP-GDH基因可以作为一种新的选择标记用于植物基因工程的研究.     

Metabolic Regulation by Homoserine in Escherichia coli B/r

A mathematical analysis of branched pathway regulation has led to the prediction of a novel homoserine control in Escherichia coli B. Experimental support for such control is presented in this paper. Homoserine, the precursor of both threonine and methionine, inhibits nicotinamide adenine dinucleotide phosphate (NADP(+))-specific glutamate dehydrogenase (EC 1.4.1.4), the enzyme catalyzing the first reaction in ammonia assimilation. Physiological and biochemical evidence for this effect are offered. Homoserine depresses the growth rate of the organism, and glutamate, the product of the inhibited reaction, reverses this effect. The NADP(+)-specific glutamate dehydrogenase activity in cell-free extracts is inhibited by homoserine, and this inhibition parallels the restriction of growth rate. These effects are found in other enteric bacteria which share a similar overall pattern of control for the amino acids derived from aspartate. On the other hand, a sampling of more distantly related species which have different pathways and/or regulatory patterns provides no evidence for homoserine inhibition of the glutamate dehydrogenase reaction.     

Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase

NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum. Isophthalate, a potent in vitro inhibitor of Aspergillus niger NADP-GDH [Noor S, Punekar NS. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology 2005;151:1409-19], was evaluated for its efficacy in vivo. Dimethyl ester of isophthalate (DMIP), but not isophthalate, inhibited A. niger growth on agar as well as in liquid culture. This was ascribed to the inability of isophthalate to enter fungal mycelia. Subsequent to DMIP addition however, intracellular isophthalate could be demonstrated. Apart from NAD-GDH, no other enzyme including NAD-glutamate synthase was inhibited by isophthalate. A cross-over at NADP-GDH step of metabolism was observed as a direct consequence of isophthalate (formed in vivo from DMIP) inhibiting this enzyme. Addition of ammonium to DMIP-treated A. niger mycelia resulted in intensive vacuolation, retraction of cytoplasm and autolysis. Taken together, these results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation.     

α-Ketoglutarate biosynthesis in wild and industrial strains of Lactococcus lactis

Aims:  This study was carried out to explore the ability of wild and industrial strains of Lactococcus lactis to produce α-ketoglutarate (α-KG), which is essential during the conversion of amino acids to flavour compounds.
Methods and Results:  Two pathways in α-KG biosynthesis were explored in strains of L. lactis isolated from dairy products, vegetables and commercial dairy starter cultures. Half of the strains efficiently converted glutamine to glutamate (Glu) and grew in Glu-free medium. Strains did not present isocitrate dehydrogenase and aconitase activities. However, half of the strains presented glutamate dehydrogenase (GDH) activity.
Conclusions:  The ability of L. lactis to synthesize either α-KG or Glu via GDH was confirmed. However, L. lactis strains were not able to biosynthesize α-KG by the citrate–isocitrate pathway. NADP-GDH activity was mainly found in strains isolated from vegetables, whereas NAD-GDH activity was mainly found in strains isolated from dairy products.
Significance and Importance of the Study:  The origin of isolation highly influenced NAD or NADP-GDH activities. These enzymatic activities may be correlated to the flavour production capacity of the different strains.     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Coregulation of oxidized nicotinamide adenine dinucleotide (phosphate) transhydrogenase and glutamate dehydrogenase activities in enteric bacteria during nitrogen limitation

The relationship between oxidized nicotinamide adenine dinucleotide (phosphate) [NAD(P)+] transhydrogenase (EC 1.6.1.1) and NAD(P)+ glutamate dehydrogenase in several enteric bacteria which differ slightly in their regulation of nitrogen metabolism was studied. Escherichia coli strain K-12 was grown on glucose and various concentrations of NH4Cl as the sole nitrogen source. In the range of 0.5 to 20 mM NH4Cl, the energy-independent transhydrogenase increased two to threefold. Comparable changes occurred in NAD(P)+-linked glutamate dehydrogenase. NH4Cl concentrations of 20 to 60 mM resulted in relatively constant specific activities for both enzymes. Higher exogenous NH4Cl, however, led to a decline in both activities. Isocitrate dehydrogenase, another potential source of cellular NADPH, was insensitive to NH4Cl limitation. Similar studies in the presence of glutamate and different exogenous NH4Cl concentrations again showed concerted effects on both enzymes. Growth on glutamate as the sole nitrogen source led to severe repression of both transhydrogenase and glutamate dehydrogenase. In Salmonella typhimurium, both enzymes were unaffected by limiting NH4Cl or growth on glutamate as the sole nitrogen source. Both were, however, repressed by growth on aspartate, a potential source of cellular glutamate. Coordinate changes in glutamate dehydrogenase and transhydrogenase were also evident in Klebsiella aerogenes, particularly under conditions in which glutamate dehydrogenase was regulated inversely to glutamate synthetase. Coordinate changes in glutamate dehydrogenase and transhydrogenase in enteric bacteria are discussed in terms of the possible involvement of the latter enzyme as a direct source of NADPH in the ammonia assimilation system.     

Ammonium regulation in Aspergillus nidulans

l-Glutamate uptake, thiourea uptake, and methylammonium uptake and the intracellular ammonium concentration were measured in wild-type and mutant cells of Aspergillus nidulans held in various concentrations of ammonium and urea. The levels of l-glutamate uptake, thiourea uptake, nitrate reductase, and hypoxanthine dehydrogenase activity are determined by the extracellular ammonium concentration. The level of methylammonium uptake is determined by the intracellular ammonium concentration. The uptake and enzyme characteristics of the ammonium-derepressed mutants, meaA8, meaB6, DER3, amrA1, xprD1, and gdhA1, are described. The gdhA mutants lack normal nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase (NADP-GDH) activity and are derepressed with respect to both external and internal ammonium. The other mutant classes are derepressed only with respect to external ammonium. The mutants meaA8, DER3, amrA1, and xprD1 have low levels of one or more of the l-glutamate, thiourea, and methylammonium uptake systems. A model for ammonium regulation in A. nidulans is put forward which suggests: (i) NADP-GDH located in the cell membrane complexes with extracellular ammonium. This first regulatory complex determines the level of l-glutamate uptake, thiourea uptake, nitrate reductase, and xanthine dehydrogenase by repression or inhibition, or both. (ii) NADP-GDH also complexes with intracellular ammonium. This second and different form of regulatory complex determines the level of methylammonium uptake by repression or inhibition, or both.     

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.     

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.