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)

The acrylamide quenching of the tryptophan fluorescence of apo and holo glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was studied. In the case of apo-GAPDH, the steady state fluorescence quenching cannot be described by the classical Stern-Volmer equation: strong cooperative quenching is observed. In the presence of Pi and/or cofactor NAD+, an inaccessible fraction appears. Cooperative quenching is partially suppressed in the presence of Pi and fully absent in the presence of NAD+. The measurements of the fluorescence lifetimes of the holo-enzyme by phasefluorometry allow the resolution of two lifetimes. The long-lived component is quenched by acrylamide, the short-lived component is not. Quenching induces a red shift of the steady state emission peak. The quenching parameters from the lifetime measurements allow the quantitative description of the steady state fluorescence quenching data. In agreement with the observations of Orstan and Gafni (Photochemistry and Photobiology, (1990) 31, 725-731), we find that acrylamide causes a slow, irreversible loss of activity and a reduction of titratable thiol groups when it acts on the apo-enzyme. This inactivation is strongly reduced in the presence of NAD+. We show that this inactivation is also slowed down by the presence of Pi, and that it is accompanied by a loss of the NAD+ binding site. Blocking the thiol groups with 5,5'-dithio-bis-(2-nitrobenzoic acid) does not lead to a protection against the irreversible inactivation by acrylamide, showing that reactions other than thiol modifications are involved in the irreversible effect. A fraction of the inactivation can be reversed by treatment with mercapto-ethanol.
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PMID:Acrylamide quenching of the fluorescence of glyceraldehyde-3-phosphate dehydrogenase: reversible and irreversible effects. 160 53

Enzyme protein fluorescence of di-furylacryloyl-glyceraldehyde-3-phosphate dehydrogenase (di-FA-GPDH:lambda max.excitation 290 nm, lambda max.emission 338 nm) is quenched about 28% on saturation with NAD+. Results of fluorometric titration of di-FA-GPDH with NAD+ suggest the presence of two tight and two loose coenzyme binding sites (Kdiss. 0.1 and 6.0 microM, respectively). Initial rates of the NAD(+)-dependent reaction of di-FA-GPDH with arsenate and phosphate and of mono-FA-GPDH with phosphate have been determined at varying coenzyme concentrations. The data suggest that binding of NAD+ at the tight sites does not activate the acyl group for its reaction with the acceptor (phosphate or arsenate). The group transfer reaction is dependent only on NAD+ binding to the loose sites, which carry the acyl group. The large difference in the NAD+ binding affinity to the two types of sites and their different effects on the group transfer reaction impart a sigmoidal shape to the rate versus [NAD+] plots. The sigmoidicity is abolished if the reactive SH groups at the unacylated sites are blocked by carboxymethylation.
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PMID:Differential binding of NAD+ to acyl glyceraldehyde-3-phosphate dehydrogenase and its role in the acyl group transfer reaction. 175 28

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.
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PMID:Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase. 176 2

A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Homogeneous distribution of phosphofructokinase in the rat liver acinus: a quantitative histochemical study. 183 3

Modification of a single arginine residue per subunit of rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase by 2,3-butanedione converts the enzyme into the form which retains 5-7% of the original activity and manifests cooperative properties that are absent in the native enzyme. It exhibits half-of-the-sites reactivity towards the natural substrate D-glyceraldehyde-3-phosphate. Titration of the modified enzyme with DTNB reveals only two instantaneously reacting SH groups, the total amount of SH groups approaching nine per tetramer. In the presence of 8 M urea, an additional seven SH groups become accessible to DTNB. This suggests that the arginine modification imposes some conformational constraints which affect the microenvironment of the active site cysteine residues in two subunits of the tetramer. The changes do not influence the interaction between the essential cysteine residue and NAD+ which is responsible for the change transfer complex formation, since the molar extinction coefficient of the apoenzyme-NAD+ complex, epsilon 360, was not altered upon the arginine modification. The native and close to four in the case of the native enzyme and about three with the modified one. The apparent pK values of Cys-149 within the functioning active centers of the tetramer were determined from the pH profiles of the inactivation rates in presence of iodoacetamide. The apparent pKa of the essential thiols was found to change upon enzyme modification from 9.44 to 10.07 in the apoenzyme and from 9.17 to 9.36 in the holoenzyme. The apparent pKa of the arginine residue determined from the pH dependence of the inactivation rate was equal to 9.0 and did not change upon apo-holo enzyme transition.
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PMID:Rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase is locked in the asymmetric state by chemical modification of a single arginine per subunit. 193 68

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.
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PMID:Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence. 203 52

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deducted from the coding gene. The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism. With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes. The phylogenetic meaning of this relationship is discussed.
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PMID:Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus. 211 59

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.
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PMID:Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli. 216 75

Glyceraldehyde-3-phosphate dehydrogenase was found to bind in vitro to purified, human erythrocyte glucose transporter reconstituted into vesicles. Mild tryptic digestion of the glucose transporter totally inactivated the binding, suggesting that the cytoplasmic domain of the transporter is involved in the binding to glyceraldehyde-3-phosphate dehydrogenase. The binding was abolished in the presence of antisera raised against the purified glucose transporter, further supporting specificity of this interaction. The binding was reversible with a dissociation constant (Kd) of 3.3 x 10(-6) M and a total capacity (Bt) of approximately 30 nmol/mg of protein indicating a stoichiometry of one enzyme-tetramer per accessible transporter. The binding was sensitive to changes in pH showing an optimum at around pH 7.0. KCl and NaCl inhibited the binding in a simple dose-dependent manner with Ki of 40 and 20 mM, respectively. The binding was also inhibited by NAD+ with an estimated Ki of 3 mM. ATP, on the other hand, enhanced the binding by up to 3-fold in a dose-dependent manner with an apparent Ka of approximately 6 mM. The binding was not affected by D-glucose or cytochalasin B. The binding did not affect either the glucose or cytochalasin B in binding affinities or the transport activity of the transporter. However, the enzyme was inactivated totally upon binding to the transporter. Based on these findings, we suggest that a significant portion of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes exists as an inactive form via an ATP-dependent, reversible association with glucose transporter, and that this association may exert regulatory intervention on nucleotide metabolism in vitro.
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PMID:An ATP-modulated specific association of glyceraldehyde-3-phosphate dehydrogenase with human erythrocyte glucose transporter. 239 33

In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.
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PMID:The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique. 241 97


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