NADP-dependent Glutamate Dehydrogenase Research Articles

Cloning of the arg-12 gene of Neurospora crassa and regulation of its transcript via cross-pathway amino acid control.

The arg-12 locus of Neurospora crassa encodes ornithine carbamoyl transferase, which is one of many amino acid synthetic enzymes whose activity is regulated through cross-pathway (or general) amino acid control. We report here the use of probes derived from the functionally equivalent arg-B gene of Aspergillus nidulans to identify and clone a 10 kb Neurospora DNA fragment carrying the arg-12 gene. Short Neurospora DNA probes derived from this fragment were used to identify a 1.5 kb polyA+ transcript of the arg-12 region. Arg-12 transcript levels increased approximately 20 fold under conditions of arginine or histidine limitation in strains having normal cross-pathway regulation (cpc-1+) but showed no such response in a cpc-1 mutant strain impaired in this regulation. Time course studies in cpc-1+ strains revealed a rapid response (within 10 m) of arg-12 transcript levels following inhibition of histidine synthesis by 3 amino 1,2,4 triazole, but a delayed response following arginine deprivation of an arginine requiring strain. In contrast to the behaviour of arg-12 mRNA, the level of the Neurospora am gene transcript (specifying NADP dependent glutamate dehydrogenase) was unaffected either by amino acid limitation or by the cpc-1 mutation. A possible role for the cpc-1+ product as a positive regulator of transcription of genes subject to cross-pathway control is discussed.

Isoenzyme variation in schizonts of Plasmodium vivax from Burma

Fifteen isolates of blood containing Plasmodium vivax were collected from hospital in-patients in Rangoon and the schizonts were harvested for starch gel electrophoresis of the following parasite isoenzymes—glucose phosphate isomerase (GPI) (EC.5.3.1.9), NADP-dependent glutamate dehydrogenase (GDH) (EC.1.4.1.4), lactate dehydrogenase (LDH) (EC.1.1.1.27) and 6-phosphogluconate dehydrogenase (6PGD) (EC.1.1.1.43). Variation was found only in GPI. The other three isoenzymes appeared to be invariant in all the isolates. Forms of GPI were recorded in two isolates of P. vivax which differed from those reported for P. falciparum; these new forms were designated GPI-4 and GPI-5.

Glutamine Cycling in Neurospora crassa

Summary: The accumulation of amino acids and the excretion of ammonium have been studied in various Neurospora crassa ammonium assimilation mutants, together with the labelling of glutamine in the presence of [U-14C]sucrose when N. crassa grows on glutamine as the nitrogen source. Ammonium coming from glutamine degradation by the ω-amidase pathway is assimilated by NADP-dependent glutamate dehydrogenase and glutamine synthetase. The operation of these enzymes results in a cycling of glutamine that seems to be essential for cell growth. In addition, glutamine is also converted to glutamate by glutamate synthase.

Glutamate dehydrogenase (NADP-dependent) mRNA in relation to enzyme synthesis in Euglena gracilis. Evidence for post-transcriptional control.

Cells of Euglena gracilis Klebs strain z Pringsheim had high NADP-dependent glutamate dehydrogenase activity when grown on glutamate as nitrogen source but activity was completely repressed in cells grown on ammonium (NH4+). A 120-fold purification of NADPH-glutamate dehydrogenase (subunit Mr = 45 000) was achieved from glutamate-grown cells by affinity chromatography on blue Sepharose CL-6B. Antisera raised against the homogeneously pure protein were used to demonstrate that increase in NADPH-glutamate dehydrogenase activity on transfer from NH4+ to glutamate medium resulted from an increase in the amount of protein. Glutamate NH4+-grown cells were labelled with L-[35S]methionine and anti-(NADPH-glutamate dehydrogenase) used to immunoprecipitate the dehydrogenase from cell extracts. NADPH-glutamate dehydrogenase protein was detected in glutamate-grown but not NH4+-grown cells. Anti-(NADPH-glutamate dehydrogenase) was used to detect NADPH-glutamate dehydrogenase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in glutamate NH4+-grown cells, there being no apparent difference in mRNA abundance between cells showing a tenfold difference in NADPH-glutamate dehydrogenase specific activity. These results indicate that the synthesis of this dehydrogenase is regulated primarily at the post-transcriptional level.

Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase.

The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa.

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on L-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on L-glutamate, L-proline, or L-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru- phenotype of the mutants.

Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae

The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described. Two DNA fragments selected from a yeast genomic library in phage λgt11were shown by restriction analysis to share 2.5 kb of common sequence. A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh − strain of S. cerevisiae and directs substantial overproduction of NADP-GDH. One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N. crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.

Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation

The ‘high ammonia pathway’ enzyme glutamate dehydrogenase (NADP +) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth in reached. Purified glutamate dehydrogenase (NADP +) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP +) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP +) inactivation is caused or followed by rapid proteolysis.

Regulation of the Key Enzymes of Methylated Amine Metabolism in Candida boidinii

Nitrogen assimilation during growth of Candida boidinii on methylated amines as sole nitrogen source involves NADP-dependent glutamate dehydrogenase. Changes in enzyme activities during the adaptation of the yeast from growth on ammonium to growth on trimethylamine were examined. No ammonia, dimethylamine or monomethylamine could be detected in the medium during growth on trimethylamine. When two methylated amines were supplied together, they were used simultaneously, although monomethylamine was metabolized more quickly than the others. When cells were grown on a low concentration of ammonium plus higher concentrations of di- or trimethylamine, the ammonium was used first. NADP-dependent glutamate dehydrogenase was the first enzyme to be derepressed, followed by methylamine oxidase and formaldehyde dehydrogenase. Di- and trimethylamine mono-oxygenase activities only appeared when the ammonium concentration fell below 0.5 mM. At this point amine utilization could be detected and no diauxic lag was observed in the growth curve. During growth on limiting ammonium, there was an increase in the activity of methylamine oxidase (150-fold) and catalase (5-fold) in the absence of any amine, but no amine mono-oxygenase activity was detected. Addition of ammonium ions to cultures growing on dimethylamine produced an immediate repression of synthesis of methylamine oxidase, NADP-dependent glutamate dehydrogenase and the two amine mono-oxygenases. An inverse correlation was found between intracellular ammonium concentration and methylamine oxidase activity. Ammonium ions also inhibited the uptake of dimethylamine or trimethylamine by washed suspensions of dimethylamine-grown cells. It is concluded that the control of methylamine oxidase and catalase and (independently) of NADP-dependent glutamate dehydrogenase is by repression of enzyme synthesis by ammonium, while expression of amine mono-oxygenases seems to require the amine to be present in the medium. Formaldehyde and formate dehydrogenases seem also to be induced by their respective substrates.

Evidence for the participation of NADP-Glutamate dehydrogenase in the ammonium assimulation of Chlorella sorokiniana

The addition of ammonium to Chlorella sorokiniana growing on nitrate results in the de novo synthesis of two isoenzymes of the NADP-dependent glutamate dehydrogenase. This can be obtained by the inhibition of glutamine synthetase with methionine sulfoxime (MSX). The K m-value for ammonium of this NADP-dependent glutamate dehydrogenase was 50 μM in the presence of low ammonium concentrations and nearly 25-fold higher with high ammonium concentrations. These values of K m were 10 3-fold lower than usually reported. The effective role of this NADP-dependent glutamate dehydrogenase in the nitrogen metabolism and its regulation with respect to glutamine synthetase is discussed.

NADP-dependent glutamate dehydrogenase from a facultative methylotroph, Pseudomonas sp. strain AM1.

The NADP-dependent glutamate dehydrogenase (EC 1.4.1.4.) elaborated by the methylotrophic bacterium Pseudomonas sp. strain AM1 when growing on succinate and ammonium chloride was studied. The enzyme, which has a pH optimum of 9.0, was purified 140-fold and shown to have Km values of 20.2 mM, 0.76 mM, 0.033 mM, and 31.6 mM for ammonia, alpha-ketoglutarate, NADPH, and glutamate, respectively. The native molecular weight was determined by polyacrylamide gel electrophoresis to be 190,000, and electrophoresis under denaturing conditions in the presence of sodium dodecyl sulfate revealed a minimum molecular weight of 50,000. The enzyme was highly specific; NADH was unable to replace NADPH in the reaction, various alpha-keto acids could not replace alpha-ketoglutarate, and neither methylamine nor hydroxylamine could substitute for ammonia. Glutamate dehydrogenase was synthesized by the bacteria only when ammonia was its nitrogen source and was repressed if methylamine or nitrate were provided as sources of nitrogen instead of ammonia.

Glutamate dehydrogenase from wild type Neurospora crassa; inactivation by photo-oxidation

The NADP dependent glutamate dehydrogenase from wild type Neurospora crassa is inactivated by exposure to light in the presence of the dye, Methylene Blue. 1. 2. Photo-oxidation appears to disturb the conformational equilibrium which controls the activity of this enzyme. 2. 3. Data obtained suggests that the modified group is the same as that reactive to the histicline reagent, diethylpyrocarbonate.

Isoenzyme characterization of chloroquine-resistant isolates of Plasmodium falciparum from Burma

Sixty isolates of Plasmodium falciparum collected from hospital in-patients in Rangoon and out-patients from villages near Rangoon were tested in vitro for chloroquine sensitivity and then cultured to carry out starch gel electrophoresis of the following parasite enzyme: glucose phosphate isomerase (GPI) (EC.5.3.1.9.), NADP-dependent glutamate dehydrogenase (GDH) EC.1.4.1.4), lactate dehydrogenase (LDH) (EC.1.1.1.27) and 6-phosphogluconate dehydrogenase (6PGD) (E.C.I.1.1.44). Variations were observed in three (GPI, GDH and LDH) of the four enzymes examined. Three forms of parasite GPI, two forms of parasite GDH, two forms of parasite LDH and only one form of parasite 6PGD were identified. Enzyme polymorphism was more common in isolates with higher resistance to chloroquine. One isolate with parasite GDH-2 and six isolates with parasite LDH-2 were recorded from chloroquine-resistant cases.

The sub-cellular localisation of pyruvate carboxylase and of some other enzymes in Aspergillus nidulans.

The sub-cellular localisation of enzymes has been defined by latency analysis, and fractionation by differential centrifugation, in cell-free extracts prepared from the mycelium of Aspergillus nidulans by growth in the presence of 2-deoxyglucose followed by treatment with a mixture of beta-glucuronidase, sulphatase and beta-glucanase and exposure to N2 cavitation at 5.2 PMa. In such extracts pyruvate carboxylase and NAD-dependent and NADP-dependent glutamate dehydrogenases are exclusively localised in the cytosol whereas all the other enzymes studied have sub-cellular localisation patterns similar to those described for mammalian liver. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for many of the enzymes, e.g. NAD--malate dehydrogenase, NADP--isocitrate dehydrogenase, glutamate/oxaloacetate transaminase, fumarase, which show a marked extent of incomplete latency and the presence of significant activity in the mitochondrial and cytosolic fractions prepared by differential centrifugation. A novel method is described for detection of citrate synthase activity following electrophoresis of the cell-free extract. Application of this method confirms the absence of a unique cytosolic isoenzyme of citrate synthase and hence shows that citrate synthase activity detected in the soluble fraction results from damage to the mitochondria during isolation. A scheme is proposed on the basis of these data to describe the organisation of lipid and amino acid synthesis from glucose in an organism which possesses a cytosolic pyruvate carboxylase.

Yeast 3-Hydroxy-3-methylglutaryl-CoA Reductase: an Enzyme Committed to Catabolite Derepression Before Exhaustion of Fermentable Substrate

SUMMARY: Saccharomyces cerevisiae grown on glucose (1 %, w/v) medium exhibited diauxic growth because of the transition from catabolite-repressed fermentative metabolism to derepressed oxidative metabolism. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) was object to catabolite repression and required cytoplasmic protein synthesis for derepression. The addition of glucose (2 %, w/v) at up to 4 h prior to glucose exhaustion had no effect on subsequent derepression of HMG-CoA reductase, suggesting that the enzyme was irreversibly committed to derepression at this stage. However, other derepressible enzyme activities such as NAD-dependent glutamate dehydrogenase (EC 1.4.1.2) and malate dehydrogenase (EC 1.1.1.37) did not show a commitment to derepression at this time. In contrast, NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) was induced by the addition of glucose.

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of 14CO2 from 14C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.

Analysis of proteinase A function in yeast.

Yeast mutants lacking proteinase A were isolated. One of these mutants (Hb I) is characterized in detail. The mutation called pra1 segregates 2:2 in meiotic tetrads indicating a single gene mutation. No anti-(proteinase A) cross-reacting material can be detected. Diploids heterozygous for pra1 show gene dosage. Thus, it appears that PRA1 might be the structural gene for proteinase A. Results obtained with this mutant show that proteinase A is not a vital component of the vegetative cell cycle. The mutant exhibits normal mitotic growth under rich and poor growth conditions and shows normal mating. Enzymes subject to carbon catabolite inactivation and inactivation of NADP-dependent glutamate dehydrogenase, processes which were proposed to be of proteolytic nature, are not affected by the absence of proteinase A. However, protein degradation under sporulation conditions is about 30% reduced in proteinase A mutant cells. The differentiation process of sporulation is also disturbed leading to a 40% reduced sporulation frequency in mutant cells.

Electrophoretic studies on three enzymes from Sarcocystis species in sheep

Thin-layer starch gel electrophoresis was used to distinguish isoenzymic differences between three species of Sarcocystis from sheep, Sarcocystis tenella, S. gigantea and S. medusiformis. Three enzymes, NADP dependent glutamate dehydrogenase, phosphoglucomutase and 6-phosphogluconate dehydrogenase showed distinctive enzyme banding patterns for the three species. ac]19800301

Nitrogen control in Pseudomonas aeruginosa: a role for glutamine in the regulations of the synthesis of nadp-dependent glutamate dehydrogenase, urease and histidase.

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.

Sequential production of enzymes and basidiospore formation in fruiting bodies of Coprinus macrorhizus

The production of NADP-dependent glutamate dehydrogenase was initiated at the stage of first meiotic prophase in pileus cells but not in stipe cells of dikaryotic and monokaryotic fruiting bodies in Coprinus macrorhizus. The production of chitinase and glucanases assayed with laminarin and lichenan was observed after the completion of meiosis only in pileus cells. The light conditions that were effective for the delay or inhibition of cellular events in the pileus cells were also effective for the delay or inhibition of enzyme production. But all sporeless mutants tested, which were defective at the various stages of basidiospore formation, produced the normal levels of these enzymes. The results indicate that the sequential production of enzymes and cellular events leading to basidiospore formation in pileus cells are independent from each other.