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)

We have previously shown that the inflammatory mediator interleukin-1 suppressed transcription of CYP1A1 and CYP1A2 mRNAs (Barker, C.W., Fagan, J.B., and Pasco, D.S. (1992) J. Biol. Chem. 267, 8050-8055). Since many of the actions of inflammatory mediators are mimicked by oxidative stress, we treated isolated hepatocytes with 0.25-1.0 mM H2O2 to determine whether expression of these genes is also modulated by oxidative stress. Inducer-dependent accumulation of CYP1A1 and CYP1A2 mRNAs were maximally reduced approximately 50 and 70%, respectively, by 1.0 mM H2O2. Run-on transcription analysis suggested that the effect of H2O2 was mediated transcriptionally. The reduction in CYP1A mRNA levels was not due to a reduction in the levels of all mRNAs due to some general toxic effect since H2O2 did not reduce glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, beta-fibrinogen, or albumin mRNA levels, and did not increase lactate dehydrogenase released into the medium. Insulin-mimicked H2O2 action, reducing the expression of both mRNAs, and N-acetylcysteine, which increases intracellular glutathione levels, completely reversed the insulin effect on both mRNAs and the H2O2 effect on CYP1A1 mRNA, but only partially reversed the H2O2 effect on CYP1A2 mRNA. This study indicates that the CYP1A1 and CYP1A2 genes are responsive to oxidative stress and that the majority of this responsiveness can be modified by cellular redox potential.
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PMID:Down-regulation of P4501A1 and P4501A2 mRNA expression in isolated hepatocytes by oxidative stress. 830 54

Oxidative stress is known to cause cataracts in lens culture systems and is believed to be an important factor contributing to human cataracts. In this communication, it is demonstrated that cataract development of cultured rat lenses produced as a result of photochemically induced oxidation in a 4% oxygen atmosphere similar to the native environment of the lens can be blocked by the transition metal complex AL-3823A. In this system, riboflavin is added to the medium as a photosensitizer. AL-3823A acts primarily as a glutathione peroxidase mimic, which catalytically metabolizes H2O2 and also has low superoxide dismutase-like activity. Measurements of H2O2, O2.-, and OH. indicate that appreciable levels of the first two of these oxidants and low levels of OH. are produced by this photochemical stressing system. The H2O2 concentrations are similar to those found in some patients with cataracts. The development of cataracts was followed over a 96-hr period. Transparency, hydration, glyceraldehyde-3-phosphate dehydrogenase activity, and protein and nonprotein thiol were monitored. All parameters show marked changes during the 96-hr period. However, in the presence of 200 microM AL-3823A, no difference between control and light-exposed lenses was observed with respect to these parameters. The results suggest that in vivo human cataract development caused by oxidative stress may be prevented by compounds of this type.
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PMID:Photochemically induced cataracts in rat lenses can be prevented by AL-3823A, a glutathione peroxidase mimic. 835 43

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

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.
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PMID:Characterization of manganese(II) binding site mutants of manganese peroxidase. 868 36

Evidence of in vivo oxidant-induced injury in inflammatory bowel disease (IBD) is largely indirect. Colon epithelial crypt cells (CEC) from paired specimens of histologically normal and inflamed bowel from IBD patients with active disease were examined for altered protein thiol redox status as an indicator of oxidative damage. When CEC preparations from 22 IBD patients were labeled with the reduced-thiol-specific probe [14C]-iodoacetamide (IAM), there was decreased labeling of a number of proteins indicating oxidation of thiol groups in CEC from inflamed mucosa compared to paired normal mucosa, especially the loss of thiol labeling of a 37-kD protein which was almost completely lost. The loss of reduced protein thiol status for the 37-kD band was paralleled by loss of epithelial cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) enzyme activity, an enzyme known to contain an essential reduced cysteine (Cys149) at the active site. The identity of the 37-kD protein as GADPH monomer was confirmed by NH2-terminal amino acid sequence analysis. To examine whether this type of in vivo injury could be attributed to biologically relevant oxidants produced by inflammatory cells, CEC prepared from normal mucosa were exposed to H2O2, OCl-, nitric oxide (NO), and a model chloramine molecule chloramine T (ChT) in vitro. Dose-dependent loss of IAM labeling and GAPDH enzyme activity was observed. The efficacy (IC50) against IAM labeling was OCl- >> ChT > H2O2 > NO (52 +/- 3, 250 +/- 17, 420 +/- 12, 779 +/- 120 microM oxidant) and OCl- >> ChT > NO > H2O2 (89 +/- 17, 256 +/- 11, 407 +/- 105, 457 +/- 75 microM oxidant), respectively, for GAPDH enzyme activity. This study provides direct evidence of in vivo oxidant injury in CEC from inflamed mucosa of IBD patients. Oxidation and inhibition of essential protein function by inflammatory cells is a potential mechanism of tissue injury that may contribute to the pathogenesis of the disease and supports the exploration of compounds with antioxidant activity as new therapies for IBD.
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PMID:Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. 869 Jul 84

H2O2 and free radical-mediated oxidative stresses have been implicated in mediating amyloid beta (1-40) [A beta (1-40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2-scavenging enzyme catalase protects neurons in culture against A beta-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. A beta-mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection. Three intracellular biochemical markers of H2O2-mediated oxidative stress were unchanged by A beta treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. lonspray mass spectra of A beta in the incubation medium indicated that A beta itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to A beta. Our results challenge a pivotal role for H2O2 generation in mediating A beta toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.
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PMID:Amyloid beta-mediated oxidative and metabolic stress in rat cortical neurons: no direct evidence for a role for H2O2 generation. 885 44

13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H2O2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 +/- 2 and 41 +/- 3%). However, with H2O2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 +/- 2%) than glucose (9 +/- 1%). Also, when [2-(13)C] glucose was supplied, accumulation of [2-(13)C] and [5-(13)C] fructose 1,6-bisphosphate was observed in the presence of H2O2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H2O2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.
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PMID:Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts. 890 92

Ozone is a widespread component of polluted air. It is the cause of many adverse effects on the lung such as decreased athletic performance and exacerbation of asthma. Ozone inactivated acetylcholine esterase (AChE) both in intact washed human erythrocytes and in ghosts prepared from the erythrocytes. This is consistent (a) with the location of AChE on the outer face of the membrane and (b) with the change in structure of AChE when amino acids were oxidized. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) of intact washed erythrocytes was unaffected by ozone. However, ozone severely inactivated G3PDH of ghosts, much more severely than AChE in ghosts. This result raised questions about the relative permeability of intact erythrocytes and ghosts and also about the inherent susceptibility of the two enzymes. Inhibition of the ozone-treated erythrocyte AChE with the competitive inhibitor trimethyl-(p-aminophenyl) ammonium chloride was measured. The inhibited enzyme had a higher K(M) and slightly lower Vmax than the control. Ozone did not affect the K(M) of the uninhibited enzyme but decreased the K(M) of the inhibited enzyme. Ozone decreased the Vmax of both the inhibited and the uninhibited enzyme. The K(I) was unchanged by the treatment with ozone. This suggested that the active site of the enzyme was not affected by ozone, but other features of the protein were changed by ozone. The effects of products of lipid ozonolysis [hydrogen peroxide, nonanal, and 1-palmitoyl-2-(9-oxononanyl)-sn-3-glycerophosphorylcholine (PN1PC)] were tested on the ghost preparations. The ozonolysis products were tested at concentrations equivalent to calculated amounts that could have been produced by ozone. Hydrogen peroxide had no effect on the G3PDH and AChE. Nonanal slightly increased the permeability of the ghost membrane, as judged by the increase in rate of G3PDH in the absence of Triton X-100, but did not inhibit enzyme activity. PN1PC increased the permeability of the ghosts, as judged by the increase in rate of G3PDH in the absence of Triton X-100. There was also an increase in the activity of G3PDH in the presence of Triton X-100. AChE was not inhibited by ozone in the presence or absence of Triton X-100.
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PMID:Reaction of ozone with enzymes of erythrocyte membranes. 891 45

A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.
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PMID:Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and coordination. 910 22

The redox homeostasis is controlled by several enzyme systems. Sulfhydryl groups in lens proteins are very sensitive to oxidative stress and can easily conjugate with nonprotein thiols (S-thiolation) to form protein-thiol mixed disulfides. We have observed an elevation of protein S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) in cataractous lenses from humans and from animal models subjected to oxidative stress. We also observed that these protein-thiol mixed disulfides could be spontaneously dissociated and lowered to basal levels if the lens which was pre-exposed to H2O2 was subsequently cultured in H2O2-free medium. This suggests that the lens has a system to repair oxidative damage through dethiolation thereby restoring its redox homeostasis. In other tissues, an enzyme, thioltransferase (TTase), has been shown to be responsible for thiol/disulfide regulation. We recently demonstrated the presence of this enzyme in the lens and in cultured lens epithelial cells. Here, we investigated the response of TTase to H2O2 stress and its possible repair function in cultured lens epithelial cells. Rabbit lens epithelial cell line N/N 1003A was raised to confluence, trypsinized and plated at 0.8 million cells per 60 mm culture dish. The cells were incubated overnight in Eagle's minimum essential medium (MEM) with 1% rabbit serum and then in serum-free MEM for 30 min before a bolus of 0.5 mm H2O2 was added. At intervals of 5, 15, 30 min and up to 3 hr, the cells were harvested and used for enzyme assays for TTase, glutathione reductase (GR), glutathione peroxidase (GPx) and glyceraldehyde-3-phosphate dehydrogenase (G-3PD). Free GSH, total SH and PSSG and PSSC were also determined. Hydrogen peroxide in the medium was measured at each time point. Cells incubated without H2O2 were used as controls. The results showed that the H2O2 concentration was reduced to 50% within 30 min and was undetectable at 2 hr. Cellular GSH dropped to 40% within 5 min and stayed at this level before it began to increase at 90 min and completely recovered by 2 hr. The total SH groups were similar to free GSH. PSSG and PSSC increased 6.5 and 2 times respectively before 30 min and then decreased when GSH started to recover. G-3PD was most sensitive to H2O2 and lost 95% activity within 5 min. The activity was regained quickly when H2O2 diminished in the medium. A similar but less severe pattern was observed in both GPx (60% loss at 60 min) and GR (30% loss at 90 min). In contrast, TTase activity remained constant during the entire 3 hr. Only when a higher dose of H2O2 (0.8-1.0 mM) was used, did TTase activity show a brief loss (<30% at 60 min) and a swift recovery. Cells exposed to H2O2 exhibited a normal morphology with no evidence of DNA fragmentation. The lens epithelial cells showed a remarkable ability to repair the early damages induced by H2O2. The unusual oxidative stress-resistant property displayed by TTase, coupled with its known function suggest that it plays an important role in the repair of oxidative damage.
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PMID:Thioltransferase is present in the lens epithelial cells as a highly oxidative stress-resistant enzyme. 959 40


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