Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Controlled oxidation of rat muscle glyceraldehyde-3-phosphate dehydrogenase (GPDH) was carried out in an attempt to stimulate age-related effects observed in enzyme samples purified from old animals. A comparative study of the "simulated aged" and of native young and old GPDH forms was done using fluorescence techniques. The present work is based on our previous findings that the locus of the age-related modifications in GPDH is in the nicotinamide-binding site, where the catalytically active Cys-149 residue is located, and that an increase in oxidation potential occurs in old animal tissues which may enable various oxidizing agents to play a significant role in the inactivation of certain enzymes. Thus it has been suggested that the loss of specific activity observed in old GPDH may be due to subtle and irreversible conformational changes caused by reaction of Cys-149 with these agents. The circularly polarized luminescence (CPL) spectrum emitted by the fluorescent sulfhydryl reagent I-AEDANS covalently bound to GPDH through Cys-149 at the nicotinamide binding site, revealed a significant difference in conformation between these sites in young and old GPDH forms. Large differences were also observed between corresponding spectra when the binding sites were saturated with NAD+, reflecting the development of marked conformational changes in both young and old GPDH species upon coenzyme binding. The oxidizing reagents employed in the current study (hydrogen peroxide, superoxide radical and atmospheric dioxygen) are all expected to be more commonly encountered in the less reducing environment of old animal tissues. All of them, though to a different extent, caused a significant inactivation of the enzyme dependent on the initial oxidant concentration. Although the original enzymatic activity could be partially restored by incubation with a reducing agent, the prior oxidation was found to induce some irreversible structural changes as expressed in a decrease in the number of fast reacting SH groups. The extent of irreversible inactivation was a function of both oxidant concentration and the duration of exposure to the oxidant. The affinity of the oxidized GPDH species (termed "aged") toward coenzyme, as monitored by fluorometric titrations, was markedly lower than that observed for both the native young and old GPDHs. In addition, the CPL spectra of the "aged" enzymes were different from those obtained for both native forms.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of aging of rat muscle glyceraldehyde-3-phosphate dehydrogenase studied by selective enzyme-oxidation. 282 73

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.
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PMID:Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules. 290 26

Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (1) resulted in time-dependent, irreversible inhibition of GAPDH. The kinetics of inactivation were biphasic, exhibiting an initial rapid phase and a slower second phase. Pentalenolactone methyl ester (2) also irreversibly inactivated GADPH, albeit at a slower rate and with a higher KI. The substrate glyceraldehyde-3-phosphate (G-3-P) afforded protection against inactivation by 1, whereas the presence of NAD+ in the incubation mixture stimulated the inactivation by increasing the apparent affinity of the enzyme for the inhibitor. In steady-state kinetic experiments, 1 acted as a competitive inhibitor of GAPDH with respect to G-3-P but exhibited uncompetitive inhibition with respect to NAD+. Inactivation of NAD+-free apo-GAPDH by 1 showed simple pseudo-first-order kinetics. By titrating the free thiol residues of partially inactivated GAPDH, it was found that both pentalenolactone and pentalenolactone methyl ester react with all four Cys-SH residues of the tetrameric GAPDH.
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PMID:Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone: kinetic and mechanistic studies. 293 Jan 99

A 1523-base-pair DNA fragment, spanning the gap gene from Escherichia coli, has been sequenced. It contains an open-reading frame whose length (330 amino acids) is in agreement with D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecular mass. This coding sequence is preceded by a Shine-Dalgarno complementary sequence and by two overlapping promoter-like structures. The codon usage within gap is consistent with that expected for a gene which is strongly expressed. The amino acid sequence of the E. coli GAPDH, deduced from the DNA sequence, contains all the amino acids postulated to play a functional role in GAPDH. Comparison of the E. coli enzyme with enzymes from other species reveals different evolutionary behaviour of the NAD+-binding domain and of the catalytic domain of GAPDH. The E. coli enzyme is found to be more similar to eucaryotic enzymes than to enzymes from thermophilic bacteria. This observation is discussed in terms of adaptation to growth at high temperature.
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PMID:Nucleotide sequence of the Escherichia coli gap gene. Different evolutionary behavior of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase. 299 Sep 26

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutants proteins--His176----Asn, Cys149----Ser, Cys149----Gly--were isolated from one or both of the enzymes. The study of the properties of these mutants has shown that Cys149 is clearly responsible for the information of a charge-transfer transition, named the Racker band, observed during the NAD+ binding to apoGAPDH. This result excludes a similarity between the Racker band and the charge-transfer transition observed following the alkylation of GAPDH by 3-chloroacetyl pyridine-adenine dinucleotide.
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PMID:Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase. 307 57

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms. 317 21

It has been shown recently that glyceraldehyde-3-phosphate dehydrogenase (GAPD) is one of the three major RNA-binding proteins of rabbit reticulocytes [Ryazanov, A. G. (1985) FEBS Lett. 192, 131-134]. It was suggested that, due to its RNA-binding capacity, GAPD can form loose dynamic complexes with polyribosomes. This communication reports that a considerable amount of GAPD activity can be found in the mono- and polyribosome fraction after sucrose gradient centrifugation of rabbit reticulocyte lysate. An increase of ionic strength, as well as the addition of exogenous RNA to the extract, result in the removal of GAPD from the complex with mono- and polyribosomes. It appears that GAPD forms the complex with polyribosomes due to the interaction with some exposed RNA regions of these structures. Although the interaction of GAPD with ribosomes is weak, it can be detected under physiological ionic conditions by the difference boundary sedimentation velocity technique. Association of GAPD with mono- and polyribosomes can be prevented by a low concentration (10 microM) of NADH, but not NAD+. A nitrocellulose filter binding assay also shows that NADH has a stronger inhibitory effect on the enzyme-RNA complex formation, as compared with NAD+. We propose that the RNA-mediated association of GAPD with mono- and polyribosomes can provide compartmentation of the energy-supplying system on these structures within the cell. This can maintain a high local concentration of ATP and GTP near the sites of protein synthesis.
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PMID:Association of glyceraldehyde-3-phosphate dehydrogenase with mono- and polyribosomes of rabbit reticulocytes. 327 18

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.
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PMID:Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid. 333 30

NADH and NADPH accelerate the 'in vitro' rate of proteolysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by elastase and other proteases, including lysosomal proteases. NAD+ and NADP+ have the opposite effect. Since there is a good correlation between proteolytic susceptibility of proteins and their 'in vivo' degradation rates, a possible role of the reduction-oxidation status in controlling the intracellular degradation of GAPDH is advanced.
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PMID:The reduction-oxidation status may influence the degradation of glyceraldehyde-3-phosphate dehydrogenase. 353 Aug 13

The tetrameric molecule of glyceraldehyde-3-phosphate dehydrogenase possesses the ability to bind fluorescent probes of cationic nature (auramine O and acridine orange) outside the active center. The rabbit skeletal muscle and yeast enzymes share some common features, e.g., the conformational non-equivalency of subunits; two subunits per tetramer can bind auramine O; conformational changes caused by the binding of adenyl mononucleotides and involving the microenvironment of auramine O binding sites; the ability to bind the cationic probe at pH values typical for the maximal activity of the enzyme in the reaction of glyceraldehyde 3-phosphate oxidation. The yeast and rabbit muscle enzymes are distinguished in terms of localization of the probe binding sites with respect to the active center and/or in the nature of conformational changes induced by NAD+ binding. It was demonstrated that nicotinamide mononucleotide may serve as a co-enzyme in glyceraldehyde 3-phosphate oxidation catalyzed by yeast dehydrogenase; this reaction in inhibited by AMP.
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PMID:[Comparative study of glyceraldehyde-3-phosphate dehydrogenases isolated from rabbit skeletal muscles and baker's yeast using cationic fluorescent probes]. 353 63


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