Mind the GAP: Purification and characterization of urea resistant GAPDH during extreme dehydration.

The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole-body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post-translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a Km value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta-N-acetylglucosamination, and cysteine nitrosylation, post-translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration-responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I50 of 7.3 M, while the I50 from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration-induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis.

Extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase attributed to the anti-staphylococcal activity of Lactobacillus plantarum USM8613.

Increasing levels of antibiotic resistance in pathogens, including Staphylococcus aureus, remains a serious problem for public health, leading to the need for better alternative antimicrobial strategies. The antimicrobial proteins produced by Lactobacillus plantarum USM8613 attributed to its anti-staphylococcal activity were identified as extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase (GADPH), both with different mechanisms of action. Extracellular transglycosylase, which contains a LysM domain, exerts a cell wall-mediated killing mechanism, while GADPH penetrates into S. aureus cells and subsequently induces the overexpression of autolysis regulators, resulting in S. aureus autolysis. Both extracellular transglycosylase and GADPH exert anti-inflammatory effects in S. aureus-infected HaCaT cells by reducing the expression and production of TLR-2, hBDs and various pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α, and IL-8). Taken together, extracellular transglycosylase and GADPH produced by L. plantarum USM8613 could potentially be applied as an alternative therapeutic agent to treat S. aureus skin infections and promote skin health.

Expression of glyceraldehyde-3-phosphate dehydrogenase from M. tuberculosis in E. coli. Purification and characteristics of the untagged recombinant enzyme.

The goal of the present work was to produce glyceraldehyde-3-phospate dehydrogenase from M. tuberculosis in E. coli cells in soluble and catalytically active form and to elaborate a method for the purification of the recombinant enzyme. The His-tagged recombinant enzyme (Mtb-GAPDH_His) was shown to be inactive and insoluble. The untagged enzyme (Mtb-GAPDH) was catalytically active and exhibited higher solubility. Mtb-GAPDH was purified from the cell extract using ammonium sulfate fractionation and ion-exchange chromatography. The presence of glycerol was necessary for isolation of Mtb-GAPDH, presumably, to facilitate folding of the recombinant enzyme. The yield of Mtb-GAPDH constituted 1.3 mg per 10 g of the cell biomass. The specific activity of the purified Mtb-GAPDH was 55 ±â€¯5 µmol NADH/min per mg protein (pH 9.0, 22 °C) that exceeded the activity of the previously described preparation of His-tagged recombinant GAPDH from M. tuberculosis that was co-expressed with GroEL/ES chaperone by approximately 5-fold. The results suggest that the folding of the recombinant GAPDH is hindered by the His-tag, which may result in the production of insoluble protein or in isolation of the preparation with decreased specific activity.

Isolation of Crithidia spp. from lesions of immunocompetent patients with suspected cutaneous leishmaniasis in Iran.

OBJECTIVE: Leishmania major has been considered as the main aetiological agent of cutaneous leishmaniasis in Iran. However, there are recent reports about the existence of Crithidia spp in cutaneous lesions in southern Iran. Therefore, this study was designed to decipher some morphological, biological and molecular aspects of this phenomenon. METHODS: Clinical isolates were obtained from 167 patients with cutaneous ulcers. A set of specific primers based on GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) gene were used to distinguish between Crithidia and Leishmania genera. For molecular analysis, Pulsed Field Gel Electrophoresis and Mi-Seq Illumina platform were applied. Then, morphological analysis and some biological features (including potential growth at 37 °C and the ability of infecting mammalian macrophages) were studied. RESULTS: In 92.8% of clinical cases, L. major was the only causative microorganism isolated; in 5.4% of cases, co-infection of L. major and Crithidia spp. was identified; and in 1.8% of lesions, only Crithidia spp. were found. CONCLUSION: We isolated Crithidia spp. from clinical samples of patients suspected of cutaneous leishmaniasis in Iran, indicating that Crithidia spp. are capable of surviving at human body temperature and infecting macrophage cells. This raises questions on the influence of this phenomenon on pathogenicity, therapeutic outcome and disease control strategies.

A novel moonlight function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme, which generates NADH in glycolysis. Our previous study revealed a novel role of exogenous GAPDH in the amelioration of lipopolysaccharide (LPS)-induced sepsis-related, severe acute lung injury (ALI) in mice. Here, we show the effect of extracellular GAPDH on the physiological functions of macrophages, which play an important role in the onset of sepsis and ALI. GAPDH has no effect on cell viability, while it strongly suppressed cell adhesion, spreading, and phagocytic function of LPS-stimulated macrophages. GAPDH treatment significantly reduced tumor necrosis factor (TNF)-α, while it induced interleukin (IL)-10 production from LPS-stimulated macrophages in a dose-dependent manner. It is noteworthy that heat inactivation of GAPDH lost its immunomodulatory activity. Correspondingly, NADH significantly inhibited TNF-α and enhanced IL-10 production with elevation of both M1/M2 macrophage markers. These data suggest that extracellular GAPDH induces intermediate M1/M2 macrophages for termination of inflammation, partly through its enzyme activity for generation of NADH. © 2018 BioFactors, 44(6):597-608, 2018.

Isolation of recombinant human untagged glyceraldehyde-3-phosphate dehydrogenase from E. coli producer strain.

The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass). The specific activity of the freshly purified hGAPDH constitutes 117 ± 5 µmol NADH/min per mg protein (pH 9.0, 22 °C), which is close to the specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase determined under the same conditions and several times exceeds the specific activity of his-tagged GAPDH preparations. The high enzymatic activity suggests that the recombinant enzyme retains its native structure. The described procedure may be useful for researchers who need a preparation of native hGAPDH without admixture of misfolded forms for their investigations.

Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli.

Chondroitinases (ChSases) are a family of polysaccharide lyases that can depolymerize high molecular weight chondroitin sulfate (CS) and dermatan sulfate (DS). In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in different cells like normal cells and cancer cells and the expression is relatively insensitive to experimental conditions, was expressed as a fusion protein with ChSase ABC I. Results showed that the expression level and enzyme activity of GAPDH-ChSase ABC I were about 2.2 and 3.0 times higher than those of ChSase ABC I. By optimization of fermentation conditions, higher productivity of ChSase ABC I was achieved as 880 ± 61 IU/g wet cell weight compared with the reported ones. The optimal temperature and pH of GAPDH-ChSase ABC I were 40 °C and 7.5, respectively. GAPDH-ChSase ABC I had a kcat/Km of 131 ± 4.1 L/µmol s and the catalytic efficiency was decreased as compared to ChSase ABC I. The relative activity of GAPDH-ChSase ABC I remained 89% after being incubated at 30 °C for 180 min and the thermostability of ChSase ABC I was enhanced by GAPDH when it was incubated at 30, 35, 40 and 45 °C.

Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.

Purification and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) from pea seeds.

Glyceraldehyde-3-phosphate dehydrogenase [GAPDH, NAD + oxidoreductase (phosphorylating) 1.2.1.12] catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate coupled with the reduction of NAD(+) to NADH. In addition to its role in glycolysis, this enzyme has numerous alternate functions, in both prokaryotes and eukaryotes. In plants, additional functions have been reported from multiple species including Pisum sativum. A recent study has identified that GAPDH may play an important role in seed ageing and programmed cell death. Despite this the existing purification protocols are almost 40 years old, and only partial characterization of the enzyme has been reported. In the current study, we report a modified method for purification of enzymatically active pea seed GAPDH along with the characterization of the enzyme. Using 2D gel electrophoresis our study also demonstrates that pea seeds contain four isoforms of NAD(+) dependent GAPDH.

Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase.

Plasmodium spp. solely rely on glycolysis for their energy needs during asexual multiplication in human RBCs, making the enzymes of this pathway potential drug targets. We have cloned, over-expressed and purified Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGapdh) for its kinetic and structural characterization. ∼ 30-40 mg pure recombinant enzyme with a specific activity of 12.6 units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH ∼ 9. Kinetic measurements gave KmNAD=0.28 ± 0.3 mM and KmG3P=0.25 ± 0.03 mM. Polyclonal antibodies raised in mice showed high specificity as was evident from their non-reactivity to rabbit muscle Gapdh. Western blot of Plasmodium yoelii cell extract showed three bands at MW ∼ 27, ∼ 37 and ∼ 51 kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the ∼ 51 kDa form was present only in the soluble fraction of the extract. Subcellular distribution of Gapdh in P. yoelii was examined using differential detergent fractionation method. Each fraction was analyzed on a two-dimensional gel and visualized by Western blotting. All four subcellular fractions (i.e., cytosol, nucleus, cytoskeleton and cell membranes) examined had Gapdh associated with them. Each fraction had multiple molecular species associated with them. Such species could arise only by multiple post-translational modifications. Structural heterogeneity observed among molecular species of PyGapdh and their diverse subcellular distribution, supports the view that Gapdh is likely to have multiple non-glycolytic functions in the parasite and could be an effective target for anti-malarial chemotherapeutics.

[Influence of gravity discharge on the content of isatin-binding proteins in mice: results of ground-based and space research under the program Bion-M №1].

Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period.

Transglutaminase 2-dependent deamidation of glyceraldehyde-3-phosphate dehydrogenase promotes trophoblastic cell fusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.

Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD(+)) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD(+), then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD(+) on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.

Glyceraldehyde-3-phosphate dehydrogenase of the parasitic nematode Haemonchus contortus binds to complement C3 and inhibits its activity.

Haemonchus contortus is an economically important gastrointestinal parasite that infects primarily sheep and goats. To survive inside the host, the parasite must overcome the host immune response. In this study, we have identified and characterized a complement-C3-binding protein (H.c-C3BP) from this parasite employing biochemical and molecular biology tools. Initially, a truncated form of the protein was isolated from the excretory-secretory products of the parasite using C3-Sepharose column that facilitated its identification by mass spectroscopy. Subsequently, the parent molecule was generated in E. coli, and sequence analysis confirmed it as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH reacted with the antiserum raised against the truncated protein, and the truncated protein reacted with anti-GAPDH antiserum. The protein inhibited complement function as measured by haemolytic assay and membrane attack complex (MAC) formation. Sera from H. contortus-infected animals reacted with GAPDH as well as the truncated form of the protein, which further lend support to protein secretion. Thus, the C3-binding property of H. contortus GAPDH is a new function, and it represents a new entity of complement-binding protein. Identification and characterization of H.c-C3BP should facilitate development of new therapeutics considering a key role of this protein in immune modulation.

Glyceraldehyde-3-phosphate dehydrogenase from Chironomidae showed differential activity towards metals.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to interact with different biomolecules and was implicated in many novel cellular activities including programmed cell death, nuclear RNA transport unrelated to the commonly known carbohydrate metabolism. We reported here the purification of GAPDH from Chironomidae larvae (Insecta, Diptera) that showed different biologic activity towards heavy metals. It was inhibited by copper, cobalt nickel, iron and lead but was activated by zinc. The GAPDH was purified by ammonium sulphate fractionation and Chelating Sepharose CL-6B chromatography followed by Blue Sepharose CL-6B chromatography. The 150-kDa tetrameric GAPDH showed optimal activity at pH 8.5 and 37°C. The multiple alignment of sequence of the Chironomidae GAPDH with other known species showed 78 - 88% identity to the conserved regions of the GADPH. Bioinformatic analysis unveils substantial N-terminal sequence similarity of GAPDH of Chironomidae larvae to mammalian GADPHs. However, the GADPH of Chironomidae larvae showed different biologic activities and cytotoxicity towards heavy metals. The GAPDH enzyme would undergo adaptive molecular changes through binding at the active site leading to higher tolerance to heavy metals.

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism.

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Immunoprotection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Lactococcus garvieae against Lactococcosis in tilapia.

In this study, the gene encoding 40 kDa GAPDH of L. garvieae was determined and overexpressed by using the Escherichia coli expression system. Analysis results indicated that the sequences of GAPDH of L. garvieae nucleotide and its amino acid are highly homologous (80.4-100%) to several products of GAPDH from L. garvieae and other Streptococcus-related bacteria. According to Western blotting results, rabbit antiserum and tilapia infection serum reacted strongly to the recombinant GAPDH protein. In another experiment, tilapia were immunized intraperitoneally with formalin-killed L. garvieae whole cells, recombinant GAPDH (50 µg fish(-1)) from L. garvieae or both. ISA 763A was used as an adjuvant for vaccine and saline was used as a negative control. The fish challenged at 4 weeks after immunization with GAPDH+WC+ISA had the highest survival rate at 100%, followed by fish immunized with WC+ISA or GAPDH+ISA, which had RPS values of 87.5% and 50%, respectively. Additionally, specific antibody responses against L. garvieae whole cells and GAPDH were based on enzyme-linked immunosorbent assay. Following 4 weeks of immunization, the specific antibody level of all vaccine groups significantly increased, except for antibody responses against L. garvieae GAPDH of those immunized with formalin-killed L. garvieae whole cells. Our results further demonstrated that GAPDH from L. garvieae protected tilapia from experimental L. garvieae infection, implying the potential use of L. garvieae GAPDH as a vaccine against L. garvieae.

Differential dissolved protein expression throughout the life cycle of Giardia lamblia.

Giardia lamblia (G. lamblia) has a simple life cycle that alternates between a cyst and a trophozoite, and this parasite is an important human and animal pathogen. To increase our understanding of the molecular basis of the G. lamblia encystment, we have analyzed the soluble proteins expressed by trophozoites and cysts extracted from feces by quantitative proteomic analysis. A total of 63 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and were categorized as cytoskeletal proteins, a cell-cycle-specific kinase, metabolic enzymes and stress resistance proteins. Importantly, we demonstrated that the expression of seven proteins differed significantly between trophozoites and cysts. In cysts, the expression of three proteins (one variable surface protein (VSP), ornithine carbamoyltransferase (OTC), ß-tubulin) increased, whereas the expression of four proteins (14-3-3 protein, α-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein disulfide isomerase 2 (PDI-2)) decreased significantly when compared with the levels of these proteins in trophozoites. The mRNA expression patterns of four of these proteins (OTC, α-tubulin, GAPDH, VSP) were similar to the expression levels of the proteins. These seven proteins appear to play an important role in the completion of the life cycle of G. lamblia.

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate Tetrahymena thermophila.

In the present study, we purified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is involved in cellular energy production and has important housekeeping functions, from the ciliate Tetrahymena thermophila using a three-step procedure. The enzyme was purified ~68 folds by ammonium sulfate precipitation, followed by two steps of column chromatography (DEAE-cellulose and Mono-S). The purified enzyme is a homotetramer with a molecular weight of ~120 kDa. Isoelectric focusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blot analysis showed a single 32-kDa band corresponding to the enzyme subunit using a monospecific polyclonal antibody against the T. thermophila GAPDH. The maximum of enzyme activity occurred at pH 8.0 and at 30-35°C. The apparent K(m) values for both NAD(+) and D-glyceraldehyde-3-phosphate were 0.102 ± 0.012 and 0.360 ± 0.018 mM, respectively. The maximal velocity (V(max)) was 39.40 ± 2.95 U/mg. The T. thermophila GAPDH is inhibited by oxidative and nitrosative stress reagents.

Glyceraldehyde-3-phosphate dehydrogenase as a quinone reductase in the suppression of 1,2-naphthoquinone protein adduct formation.

1,2-Naphthoquinone (1,2-NQ) is electrophilic, and forms covalent bonds with protein thiols, but its two-electron reduction product 1,2-dihydroxynaphthalene (1,2-NQH(2)) is not, so enzymes catalyzing the reduction with reduced pyridine nucleotides as cofactors could protect cells from electrophile-based chemical insults. To assess this possibility, we examined proteins isolated from the 9000g supernatant from mouse liver for 1,2-NQ reductase activity using an HPLC assay procedure for the hydroquinone of 1,2-NQ and Cibacron Blue 3GA column chromatography and Western blot analysis with specific antibody to determine 1,2-NQ-bound proteins. Among the proteins with high affinities for pyridine nucleotides that also inhibited 1,2-NQ-protein adduct formation in the presence of NADH, a 37-kDa protein was found and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using recombinant human GAPDH, we found that this glycolytic enzyme indeed catalyzes the two-electron reduction of 1,2-NQ accompanied by extensive NADH consumption under 20% oxygen conditions. When either 1,2-NQH(2) or 1,2-NQ was incubated with GAPDH in the presence of NADH, minimal covalent bonding to the enzyme occurred compared to that in its absence. These results indicate that GAPDH can inhibit 1,2-NQ-based electrophilic protein modification by conversion to the nonelectrophilic 1,2-NQH(2) via an NADH-dependent process.