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)

The subcellular localization of l-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A4 > LDH-A3B) with the peroxisomes comprising relatively more LDH-A3B than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal beta-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the beta-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA beta-oxidation system could be verified in a modified beta-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mm) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD+/NADH levels and maintain the flux of fatty acids through the peroxisomal beta-oxidation spiral.
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PMID:L-lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation. 863 3

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62 +/- 4.5 and 420 +/- 10.5 microM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx.molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.
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PMID:Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide. 868 18

The effects of troglitazone and pioglitazone on glucose and fatty acid metabolism were studied in hepatocytes isolated from 24-h-starved rats. These thiazolidinediones inhibited long-chain fatty acid (oleate) oxidation and produced a very oxidized mitochondrial redox state. By contrast, thiazolidinediones did not affect the rate of medium-chain fatty acid (octanoate) oxidation or the activity of mitochondrial carnitine palmitoyltransferase (CPT) I. Thiazolidinediones inhibited selectively triglyceride synthesis but not phospholipid synthesis. The combined inhibition of oleate oxidation and esterification by troglitazone was due to a noncompetitive inhibition of mitochondrial and microsomal long-chain acyl-CoA synthetase (ACS) activities. It was suggested that troglitazone must be metabolized into its sulfo-conjugate derivative in liver cells to inhibit mitochondrial and microsomal ACS activities. Thiazolidinediones inhibited glucose production from lactate/pyruvate or from alanine. Analysis of gluconeogenic metabolite concentrations suggested that troglitazone would inhibit gluconeogenesis at the level of pyruvate carboxylase and glyceraldehyde-3-phosphate dehydrogenase reactions. It was concluded that 1) at a similar concentration, troglitazone was more efficient than pioglitazone to inhibit fatty acid metabolism and gluconeogenesis and 2) the inhibition of gluconeogenesis by troglitazone could be the result of the inhibition of long-chain fatty acid oxidation (decrease in acetyl-CoA, NADH-to-NAD+, and ATP-to-ADP ratios).
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PMID:Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats. 886 61

Interactions of NAD-dependent dehydrogenases (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, and lactate dehydrogenase, LDH) with band 3 erythrocyte membrane protein and tubulin were characterized. At low ionic strength and un-saturating substrate concentrations, LDH tightly binds to tubulin and is thus inactivated. The Kd of the LDH-tubulin complex was calculated in inhibition and direct binding experiments (15.0 and 13.6 nM, respectively); the stoichiometry of the complex was 1.66 moles of tubulin dimer bound per mole of LDH tetramer. In the presence of 0.15 M NaCl, LDH does not bind to tubulin and tubulin-dependent inhibition of LDH activity is not detected. At low ionic strength, erythrocyte membranes affect both dehydrogenases similarly. GAPDH activity is completely inhibited by excess of erythrocyte membranes (or by excess of cytoplasmic fragment of band 3 protein). Under similar conditions, LDH activity was inhibited by 70% by erythrocyte membranes. In these experiments, 14.8.10(6) GAPDH tetramers or 25.6.10(6) LDH tetramers bound to one erythrocyte ghost (Kd is 0.13 and 0.6 microM, respectively). Increase in ionic strength (0.15 m NaCl) completely abolished the membrane-dependent inhibition of dehydrogenases; however, membranes still bound GAPDH and LDH. Under these conditions, the Kd for GAPDH was increased (up to 4.43 microM), whereas the number of membrane-bound enzyme molecules has not been significantly affected (0.75 nmoles of tetramer per 100 micrograms membrane protein). The Kd for LDH was not changed (0.76 microM), whereas the number of membrane-bound enzyme molecules was decreased (down to 0.48 nmoles of tetramer per 100 micrograms membrane protein). It is suggested that at low ionic strength, the "acidic tails" of band 3 protein and tubulin can interact with positively charged NAD-binding domains of both dehydrogenases thus inhibiting their activity. Increase in ionic strength reduces these interactions, decreasing the binding and inhibition of enzyme activities. At "physiological" ionic strength, catalytically active GAPDH and LDH can possibly bind to various sites of the erythrocyte membrane. This can be important in regulation of the transfer of the common cofactor (NAD/NADH) between their active sites.
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PMID:[Effect of erythrocyte membranes and tubulin on the activity of NAD-dependent dehydrogenases]. 896 25

NADH-dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-gamma. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.
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PMID:Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-gamma and aldolase C. 918 18

9-methoxy-N2-methylellipticinium acetate (MMEA) is representative of a series of quaternized ellipticine derivatives that are selectively cytotoxic to human brain tumor cell lines derived from non-neuronal (glial) cells (Acton et al, 1994). In an attempt to determine whether MMEA may exhibit toxicity to normal brain cells, we have examined the effect of the drug, in vitro, using sagittal slices of rat brain. Incubation of rat brain slices in an artificial cerebrospinal fluid medium containing MMEA resulted in dose-dependent leakage of lactate dehydrogenase (LDH) into the surrounding medium. However, other subcellular marker enzymes such as Na(+)-K+ATPase (plasma membrane), cytochrome c oxidase, isocitrate dehydrogenase, NADH-dehydrogenase (mitochondrial), N-acetylglucosaminidase, acid phosphate (lysosomal), glyceraldehyde-3-phosphate dehydrogenase and enolase (glycolytic enzymes) were unaffected even at the highest tested concentrations of MMEA (10 and 100 microM). Preincubation of slices with reserpine (1 nM) or, dopamine or serotonin-specific reuptake inhibitors abolished MMEA-induced toxicity in brain slices. Pretreatment of slices with piperonyl butoxide and metyrapone, inhibitor of cytochrome P-450, also prevented the toxicity of MMEA. Further, brain slices prepared from phenobarbital-treated rats showed enhanced sensitivity to MMEA; significant leakage of LDH was observed at MMEA concentrations as low as 1 nM. The present studies demonstrate the toxicity of MMEA in rat brain slices, in vitro, and suggest a role for brain cytochrome P-450 in the neurotoxicity of MMEA [corrected].
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PMID:In vitro neurotoxicity of the antitumor agent 9-methoxy-N2-methylellipticinium acetate (MMEA): role of brain cytochrome P-450. 921 92

A gel penetration technique, that measures the dilution undergone by protein equilibrium on a short tightly packed gel column, has been employed to determine the molecular masses of aldolase (160 kDa), glyceraldehyde-3-phosphate dehydrogenase (GPDH; 145 kDa) in the absence and presence of each other and of other proteins. The dilution factor (concentration of protein applied/concentration of protein after equilibration) was found to be inversely related to the molecular mass of the protein. In equimolar mixtures of aldolase and GPDH, 0.5-2.5 microM each, the two enzymes exhibited a common molecular mass value of 309-316 kDa. These enzymes did not undergo any self association or disassociation in this concentration range. Moreover, their molecular masses were unaffected by the presence of other proteins tested. When the concentration of one of these enzymes (aldolase or GPDH) was held constant and that of the other varied, the dilution factor of the former was decreased as the concentration of the latter was increased until it corresponded to a molecular mass of ca. 310 kDa at equimolar concentrations of the two enzymes. Further increase in the concentration of the variable enzyme had no effect. It has been suggested that aldolase and GPDH form a 1:1 complex of dissociation constant equal to or less than 5 x 10(-8) M. The complex was found to dissociate in the presence of KCl, (NH4)2SO4, ATP and NADH whereas its formation was favoured by fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, NAD+, ADP, AMP and phosphate ions.
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PMID:Interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase: molecular mass studies. 924 8

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.
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PMID:Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio. 928 77

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).
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PMID:CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants. 929 36

Incubation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with micromolar hydrogen peroxide concentrations does not alter the catalytic properties of GAPDH in the reaction of oxidative phosphorylation of glyceraldehyde-3-phosphate, but endows the enzyme with the ability to catalyze the reaction in the absence of inorganic phosphate, producing NADH and 3-phosphoglycerate. The reaction is supposed to occur as a result of intramolecular acyl transfer from Cys-149 to a sulfenic acid form of Cys-153, followed by hydrolysis of the intermediate. The 'mildly oxidized' form of the enzyme can be easily converted back to the form unable to catalyze glyceraldehyde-3-phosphate oxidation in the absence of phosphate, by the addition of thiols.
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PMID:Rabbit muscle GAPDH: non-phosphorylating dehydrogenase activity induced by hydrogen peroxide. 931 95


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