Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase Research Articles

Identification and verification of α-11 giardin-interacting protein

To identify and verify the interacting protein of α-11 giardin, so as provide the experimental evidence for studies on the α-11 giardin function. The yeast two-hybrid cDNA library of the Giardia lambia C2 strain and the bait plasmid of α-11 giardin were constructed. All proteins interacting with α-11 giardin were screened using the yeast two-hybrid system. α-11 giardin and all screened potential interacting protein genes were constructed into pBiFc-Vc-155 and pBiFc-Vn-173 plasmids, and co-transfected into the breast cancer cell line MDA-MB-231. The interactions between α-11 giardin and interacting proteins were verified using bimolecular fluorescence complementation (BiFC). The yeast two-hybrid G. lambia cDNA library which was quantified at 2.715 × 107 colony-forming units (CFU) and the bait plasmid containing α-11 giardin gene without an autoactivation activity were constructed. Following two-round positive screening with the yeast two-hybrid system, two potential proteins interacting with α-11 giardin were screened, including eukaryotic translation initiation factor 5A (EIF5A), calmodulin-dependent protein kinase (CAMKL) and nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), hypothetical protein 1 (GL50803_95880), hypothetical protein 2 (GL50803_87261) and a protein from Giardia canis virus. The α-11 giardin and EIF5A genes were transfected into the pBiFc-Vc-155 and pBiFc-Vn-173 plasmids using BiFC, and the recombinant plasmids pBiFc-Vc-155-α-11 and pBiFc-Vn-173-EIF5A were co-tranfected into MDA-MB-231 cells, which displayed green fluorescence under a microscope, indicating the interaction between α-11 giardin and EIF5A protein in cells. The yeast two-hybrid cDNA library of the G. lambia C2 strain has been successfully constructed, and six potential protein interacting with α-11 giardin have been identified, including EIF5A that interacts with α-11 giardin in cells.

Francis M. Martin.

Francis M. Martin.

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the alpha- and beta- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The beta-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the alpha-subunit holoenzyme(s). After 120 minutes, the alpha-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using (35)SO(4) and immunochemical procedures, the rate of synthesis of the alpha-subunit was shown to be greater than the beta-subunit during the first 80 minutes of induction. The alpha- and beta-subunits had different rates of degradation during the induction period (t((1/2)) = 50 versus 150 minutes, respectively) and during the deinduction period (t((1/2)) = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana

Chlorella sorokiniana cells, cultured for 12 hours in 30 millimolar ammonium medium, contained an ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzyme with subunits having a molecular weight of 53,000. In vitro translation of total cellular poly(A)(+) RNA, isolated from fully induced cells, resulted in synthesis of an NADP-GDH antigen with a molecular weight of 58,500. The 58,500 dalton antigen was processed in vitro, with a 100,000g supernatant prepared from broken fully induced Chlorella cells, to a protein with a molecular weight of 53,000. These data support the inference that the NADP-GDH subunit (M(r) = 53,000) is initially synthesized as a larger precursor protein (M(r) = 58,500). By use of a cytochemical staining procedure, dependent upon NADP-GDH catalytic activity, the holoenzyme was shown to be chloroplast-localized. An immunoelectron microscopy procedure, employing anti-NADP-GDH immunoglobulin G and Protein A-gold complex, showed that NADP-GDH antigen was absent from the nucleus but present in both the chloroplast and cytosol. Since synthesis of the enzyme can be inhibited by cycloheximide, the detection of NADP-GDH antigen in the cytosol was probably due to binding of the NADP-GDH antibody to nascent polypeptide chains of the precursor-protein being synthesized on cytosolic 80S ribosomes.

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells.

Turnover of ammonium-inducible glutamate dehydrogenase during induction and its rapid inactivation after removal of inducer from Chlorella sorokiniana cells.

When ammonia was removed from Chlorella sorokiniana cells, which contain an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), the activity of this enzyme decayed with a half-life of approximately 8 min. By use of rocket immunoelectrophoresis, indirect immunoprecipitation, and indirect immunoadsorption (coupled with pulse-chase experiments with 35S-labeled sulfate), the rapid initial loss in activity was shown to be due to enzyme inactivation rather than degradation of NADP-GDH antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained with anti-NADP-GDH immunoglobulin G showed that enzyme inactivation is accompanied by the conversion of enzyme subunits (Mr = 59,000) to a protein with a molecular weight of 118,000. Because this protein was stable during boiling and in the presence of sodium dodecyl sulfate and high concentrations of mercaptoethanol or dithiothreitol, it was tentatively assumed to be a covalently linked dimer of enzyme subunits. Pulse-chase experiments showed that total NADP-GDH antigen was subject to rapid degradation (t 1/2 = 88 min) in induced cells, and the same degradation rate was maintained after removal of ammonia from induced cells.

Regulation of initial rate of induction of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase during the cell cycle of synchronous Chlorella.

When synchronous cells of the eucaryotic microorganism Chlorella sorokiniana growing in nitrate medium were challenged to synthesize an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) at frequent intervals during the cell cycle the initial rate of induction (i.e., enzyme potential) of this enzyme increased in an approximately linear manner until the period of DNA replication (i.e., S phase). During the S phase, NADP-GDH potential exhibited a positive rate change proportional to the step increase in DNA level. The timing of this rate change was insensitive to large changes in cellular growth rate. This rate change could be blocked within the first cell cycle by specific inhibition of DNA replication with 2'-deoxyadenosine. The approximately linear increase in NADP-GDH potential and also of total cellular protein observed before and after the S phase is proposed to be a result of the increasing photosynthetic capacity of the cell during the cell cycle.

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.

Direct selective procedure for isolating Neurospora mutants defective in nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase

A procedure has been developed for isolating nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (am) mutants of Neurospora. Physiological, genetic, and enzymatic tests show that the new mutants are am alleles. Reconstruction tests and analysis of the new alleles show that the procedure yields a broad spectrum of lesions at the am locus. The isolation of am mutants by this technique appears to be related to the effect of am mutants on the control of the general permease.

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.

Regulation of Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase in Germinated Spores of Geotrichum candidum

Germinating spores of Geotrichum candidum produce only a nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase. Synthesis of glutamate dehydrogenase was repressed by the presence of ammonia, whereas urea, glutamate, or glutamine were ineffective. The enzyme was not subject to catabolite repression and was localized in the cell sap fraction. The glutamate dehydrogenase has been purified 93-fold and showed maximal activity at pH 8.2 in the forward and reverse directions. When measuring the initial reaction rate at pH 7.2, a variety of tricarboxylic acid cycle intermediates displayed additive and unidirectional activation of the reductive amination reaction and inhibition of the oxidative deamination reaction. The modulating effects were pH-dependent and diminished at alkaline pH values. Substrate inhibition exerted by alpha-ketoglutarate was strongest at neutral pH.Rate-concentration plot with respect to alpha-ketoglutarate at pH 7.2 was sigmoidal, and the enzyme was entirely inactive at substrate concentrations lower than 1 mm. In the presence of fumarate, the enzyme did not exhibit the cooperative interaction described above and was markedly activated at alpha-ketoglutarate concentrations lower than 1 mm. The tricarboxylic acid cycle intermediates released the enzyme from substrate inhibition by alpha-ketoglutarate. Double reciprocal rate-concentration plots displayed noncompetitive inhibition between succinate and NADP and a mixed noncompetitive and competitive inhibition between succinate and glutamate. The binding order of substrates in the reductive amination reaction was NADPH, alpha-ketoglutarate, and subsequently ammonia.

Allosteric Effects in Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase from Neurospora

Abstract Neurospora nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase is predominantly in an inactive form at pH 7.2, is fully active above pH 8.0, and is partially active at intermediate pH values. Between pH 7.0 and 7.4, and in the absence of NADPH, the enzyme is activated by the substrates 2-oxoglutarate and glutamate, as well as by the nonsubstrates citrate, isocitrate, succinate, malate, fumarate, oxalacetate, or ethylenediaminetetraacetate. Curves relating activation to activator or substrate concentration were found to be sigmoid, indicating cooperation between activating molecules. At lower pH values the addition of NADPH results in nearly complete inactivation. Under these conditions activation requires 2-oxoglutarate specifically; glutamate or nonsubstrate acids have no activating effect. However, 2-oxoglutarate activates even better than in the absence of NADPH. NADP+ causes some activation by itself and enhances activation by 2-oxoglutarate, glutamate, and succinate. It is concluded that the enzyme has allosteric properties, the possible regulatory significance of which is briefly discussed. Properties previously reported for certain mutant varieties of this enzyme obtained from amination-deficient (am) mutants and their partial revertants are now seen as distorted manifestations of the normal allosteric interactions reported here for the enzyme obtained from a wild type strain.