Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora.

Neurospora NADP-specific glutamate dehydrogenase that was treated with iodoacetate, iodoacetamide, or N-ethylmaleimide to block the thiol groups was cleaved with cyanogen bromide. Of the expected 10 peptides, based on a methionine content of 9 residues, 8 were obtained in pure form and 2 were handled as a mixture. The fragments ranged in size from 9 to 109 residues. In addition, there were isolated 6 peptides, produced by anomalous cleavage at the carboxyl groups of tryptophan residues, and two by hydrolysis of an aspartyl-proline bond. Preliminary separation of these peptides was accomplished by gel filtration followed by either ion-exchange chromatography of the larger peptides or by paper chromatography and paper electrophoresis of the smaller fragments. Ordering of the CNBr fragments in sequence was based upon sequences of tryptic and chymotryptic peptides obtained in another laboratory. The complete sequence of the protein is presented. The amino acid sequences of the bovine and chicken liver glutamate dehydrogenases previously determined show considerable homology with the NADP-specific enzyme of Neurospora in the NH2-terminal half of the molecule; this includes the region of the specifically reactive lysine residue and the portion of the sequence that has been implicated in coenzyme binding. Particularly striking is the fact that most of the residues conserved among the three homologous proteins would be expected to be important for conformational, rather than catalytic, effects. This implies that the conformation of the Neurospora enzyme must be similar in parts of its structure to the vertebrate enzymes but undoubtedly differs in some regards.     

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.     

Properties of the nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from Blastocladiella emersonii.

The nicotinamide adenine dinucleotide phosphate (NADP)-specific isocitrate dehydrogenase from Blastocladiella emersonii was purified. The enzyme was very unstable. Satisfactory stability was obtained in the presence of 0.2% ovalbumin. The enzyme had a molecular weight of about 100,000. It did not exhibit homotropic cooperativity for any of it substrates and was not affected by the allosteric modifiers citrate and adenosine monophosphate, diphosphate, and tri-phosphate. The substrate saturation studies showed both intercept and slope effects in Lineweaver-Burk plots. The Km values for isocitrate and NADP were found to be 20 and 10 muM, respectively. The product inhibition pattern was compatible with a random sequential reaction mechanism. The enzyme catalyzed the oxidative decarboxylation of isocitrate about six times better than the reductive carboxylation of alpha-ketoglutarate. The enzyme was inhibited by glyoxylate plus oxalacetate. Assays conducted in the presence of low Mg2+ concentrations exhibited a lag. This lag could be abolished by the addition of reduced NADP to the assay mixture.     

Circular dichroism spectra of glutamate dehydrogenase plus reduced nicotinamide adenine dinucleotide.

Circular dichroism (CD) spectra are reported for various concentrations of glutamate dehydrogenase in order to determine any role of protein aggregation on NADH-binding spectra. These CD spectra do appear to be sensitive to enzyme aggregation. These spectra raise some doubt about previous interpretation of CD spectra as direct evidence for a second NADH binding-site.     

Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. III. Inactivation by nitration of a tyrosine residue involved in coenzyme binding.

Neurospora glutamate dehydrogenase (NADP-specific) is rapidly inactivated upon reaction with tetranitromethane. This inactivation is completely prevented by the presence of coenzyme (NADP) or nicotinamide mononucleotide (NMN) but not by substrate. NADH, or 2'-monophosphoadenosine-5'-diphosphoribose. Amino acid analysis indicates that the primary effect of modification is nitration of a single residue of tyrosine per polypeptide chain. We have identified the reactive tyrosine by isolation of a single, uniquely labeled peptide after hydrolysis with trypsin followed by cleavage with cyanogen bromide. The modified residue proved to be tyrosine-168 in the linear sequence. This residue is not present in the part of the sequence that had been previously implicated as involved in the binding of the adenylate portion of the coenzyme. Both NMN and 2-monophosphoadenosine-5'-diphosphoribose act as competitive inhibitors of NADP in the oxidation of glutamate with Ki values of 4.65 x 10(-4) M and 4.30 x 10(-4) M, respectively. Thus, the specific protection afforded by NADP and NMN, but not by 2'-monophosphoadenosine-5'-diphosphoribose, indicates that tyrosine-168 is involved in binding the nicotinamide portion of the coenzyme.     

Purification and subunit structure of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Neurospora crassa.

Neurospora crassa nicotinamide adenine dinucleotide specific isocitrate dehydrogenase (EC 1.1.1.41) has been purified to homogeneity by the criteria of disc gel electrophoresis and sedimentation equilibrium. Purification of the enzyme is facilitated by the presence of phenylmethanesulfonyl fluoride and by the use of a ribose-linked adenosine 5'-monophosphate affinity column. The enzyme appears to be composed of nonidentical subunits of molecular weights 42 800 and 38 300 as estimated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate. From the intensity of each band and the native molecular weight, it is concluded that the enzyme is composed of either six or eight subunits, three or four of each type, respectively. The availability of pure enzyme will allow clarification of the structure of the enzyme by ligand binding studies.     

A spectrophotometric procedure for measuring oxoglutarate and determining aminotransferase activities using nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase from algae

A new spectrophotometric procedure is described for determining glutamate-dependent activities of aspartate aminotransferase, alanine aminotransferase, and ornithine aminotransferase with NADPH-linked glutamate dehydrogenase (GDH) from nitrate-grown Stichococcus bacillaris. The algal NADPH-GDH is highly specific for oxoglutarate and can catalyze the reduction of this keto acid in the presence of high glutamate concentrations, and thus is suitable for the measurement of oxoglutarate produced in glutamate-dependent amino-transferase reactions. The alga produces large amounts of NADPH-GDH which can be adequately purified in a few simple steps. The purified enzyme can be stored at 4 degrees C for several weeks without any detectable loss of activity. The algal NADPH-GDH can also be used for the estimation of small amounts of oxoglutarate in aqueous extracts.     

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.     

Pulse fluorimetry study of beef liver glutamate dehydrogenase reduced nicotinamide adenine dinucleotide phosphate complexes.

Single photon counting pulse fluorimetry has been used in order to study the two ternary complexes GDH-GTP-NADPH and GDH-L-glutamate-NADPH and the quaternary complex GDH-GTP-L-glutamate-NADPH. The fluorescence decay of the enzyme-bound NADPH is not monoexponential in any of these complexes. Moreover, it does not seem to be dependent on the coenzyme concentration. The experimental curves can be satisfactorily fitted with the sum of two exponentials, the relative amplitudes of which significantly depend on the complex studied. Thus, for dihydronicotinamide two possible environments might exist in the enzyme active sites. It is also shown that the fluorescence decay times of the enzyme are shortened by the bound NADPH.     

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.     

Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase.     

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.     

Interaction of reduced nicotinamide adenine dinucleotide with beef heart s-malate dehydrogenase.

The interaction of NADH with s-malate dehydrogenase isolated from beef heart was studied in 20 mM potassium phosphate (pH 6.9)-1 mM EDTA, with forced dialysis, fluorescence, and temperature-jump techniques. Measurements of the change in fluorescence of NADH when it is titrated with enzyme indicate NADH bound to monomeric and dimeric enzyme have different fluorescence yields. These data and the results of direct binding studies can be explained in terms of a model in which the NADH binding sites on dimeric enzyme are equivalent or nearly equivalent, and NADH binding to monomeric enzyme occurs with an affinity very similar to that of the dimer. However, the fluorescence enhancement of NADH on binding to the enzyme is different for the monomer and for each of the two dimer sites.