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Background: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) is a unique enzyme that, besides its main function in glycolysis (catalysis of glyceraldehyde-3-phosphate oxidation), possesses a number of non-glycolytic activities. The present review summarizes information on the role of oxidative stress in the regulation of the enzymatic activity as well as non-glycolytic functions of GAPDH. Methods: Based on the analysis of literature data and the results obtained in our research group, mechanisms of the regulation of GAPDH functions through the oxidation of the sulfhydryl groups in the active site of the enzyme have been suggested. Results: Mechanism of GAPDH oxidation includes consecutive oxidation of the catalytic Cysteine (Cys150) into sulfenic, sulfinic, and sulfonic acid derivatives, resulting in the complete inactivation of the enzyme. The cysteine sulfenic acid reacts with reduced glutathione (GSH) to form a mixed disulfide (S-glutathionylated GAPDH) that further reacts with Cys154 yielding the disulfide bond in the active site of the enzyme. In contrast to the sulfinic and sulfonic acids, the mixed disulfide and the intramolecular disulfide bond are reversible oxidation products that can be reduced in the presence of GSH or thioredoxin. Conclusion: Oxidation of sulfhydryl groups in the active site of GAPDH is unavoidable due to the enhanced reactivity of Cys150. The irreversible oxidation of Cys150 is prevented by Sglutathionylation and disulfide bonding with Cys154. The oxidation/reduction of the sulfhydryl groups in the active site of GAPDH can be used for regulation of glycolysis and numerous side activities of this enzyme including the induction of apoptosis.

Background: Previously, it was demonstrated that quinoid pigments of sea urchins exhibit antioxidant properties. Objective: The work presents a comparative investigation of the antioxidant and antiaggregant action of polyhydroxy- 1,4-naphthoquinones: echinochrome A, spinochrome B, spinochrome C, spinochrome E, echinamine B, histochrome. The glycolytic enzyme glyceraldehyde-3-phospate dehydrogenase (GAPDH) was chosen as a possible natural target for the action of the quinones. GAPDH is easily affected by reaction oxygen species and prone to amyloid aggregation. Methods: Influence of polyhydroxy-1,4-naphthoquinones on the enzymatic activity of GAPDH, on the oxidation of the enzyme by hydrogen peroxide as well as on its thermal aggregation and some thermodynamic parameters of the enzyme were investigated. Results: It was demonstrated that spinochromes B and C decelerate the oxidation of GAPDH by hydrogen peroxide, but do not affect the GAPDH activity in the absence of H2O2. Echinichrome A and histochrome decelerate the oxidation of GAPDH by hydrogen peroxide and moderately inhibit the activity of GAPDH. Echinamine B and spinochrome E were shown to decrease significantly the activity of GAPDH. Spinochrome E exhibits a pronounced antiaggregant action, completely suppressing thermal aggregation of GAPDH. Echinochrome A and the spinochromes prevent chaperone hsp70 from blocking by oxidized and denatured forms of GAPDH. Conclusion: Most of the quinoid pigments of sea urchins, as well as the derived preparation histochrome exhibit antioxidant action and prevent interaction of denatured proteins with each other and with chaperones. However, echinamine B and spinochrome E inhibit GAPDH, which is in agreement with previously described cytotoxic action of some polyhydroxy-1,4-naphthoquinones.

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen–polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen–polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen–polyelectrolyte complex was less damaging, suggesting that binding to the antigen–polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain. Copyright 2000 Academic Press.

Monoclonal antibodies of two clones reacting with the nonnative forms of d-glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 (GAPDH), were obtained. Antibodies of clone 6C5 belonged to IgG1 subtype; antibodies of clone 6G7 belonged to IgM type. The interaction of antibodies of both clones with the immobilized and soluble enzyme was studied. The specificity of antibodies to the definite oligomeric forms was demonstrated on immobilized monomers, dimers, and tetramers of GAPDH. The affinity of antibodies to monomeric and dimeric forms of GAPDH, either active or not, was demonstrated. At the same time the antibodies did not react with the tetrameric enzyme. The binding of antibodies had no influence on the enzymatic activity. However, the addition of antibodies to the denatured enzyme blocked the spontaneous renaturation of GAPDH. The immobilized antibodies of both clones were successfully used for the purification of GAPDH solution from the denatured admixtures.

The hydrogen peroxide‐induced ‘non‐phosphorylating’ activity of D‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) is shown to be a result of the successive action of two forms of the enzyme subunits: one catalyzing production of 1,3‐bisphosphoglycerate, and the other performing its hydrolytic decomposition. The latter form is produced by mild oxidation of GAPDH in the presence of a low hydrogen peroxide concentration when essential Cys‐149 is oxidized to the sulfenate derivative. The results obtained with a C153S mutant of Bacillus stearothermophilusGAPDH rule out the possibility that intrasubunit acyl transfer between Cys‐149 and a sulfenic form of Cys‐153 is required for the ‘non‐phosphorylating’ activity of the enzyme.

The binding of denatured B. stearothermophilusD-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the E. colichaperonin GroEL was investigated in two systems: (1) GroEL immobilized on Sepharose via a single subunit was titrated with urea-denatured soluble GAPDH and (2) a Sepharose-bound denatured GAPDH monomer was titrated with soluble GroEL. Similar apparent KDvalues for the complex GroEL·GAPDH were obtained in both cases (0.04 and 0.03 μM, respectively), the stoichiometry being 1.0 mol chaperonin per GAPDH subunit in the system with the immobilized GroEL and 0.2 mol chaperonin per Sepharose-bound GAPDH monomer. Addition of GroEL and Mg·ATP to a reactivation mixture increased the yield of reactivation of both E. coliand B. stearothermophilusGAPDHs. Incubation of the Sepharose-bound catalytically active tetrameric and dimeric GAPDH forms with the protein fraction of a wild-type E. colicell extract resulted in the binding of GroEL to the dimer and no interaction with the tetrameric form. These data suggest that GroEL may be capable of interacting with the interdimeric contact regions of the folded GAPDH dimers.

Data on alterations of the properties of glyceraldehyde-3-phosphate dehydrogenase upon oxidation of its functional groups are reviewed; a mechanism of uncoupling of oxidation and phosphorylation in glycolysis is considered. Possible ways of regulating uncoupling, and the physiological importance of this process, are discussed.

The ability ofd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH·2,3-DPGM and GAPGH·3-PGK complexes were detected. TheKdvalues of the complexes determined with purified enzyme preparations were in the range 2.40–2.48 μmfor both the GAPDH·2,3-DPGM and GAPGH·3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, theKdfor the GAPDH·2,3-DPGM complex was found to be 0.54 μm, and theKdfor the GAPDH·3-PGK complex was 0.49 μm. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein–protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

Rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase was shown to serve as a substrate for Ca 2+/calmodulin-dependent protein kinase II with a Kmof 0.33 μM and a Vmaxof 2.63 μmol · min −1· mg −1at pH 7.5 and 30°C. In the absence of calmodulin, the Vmaxwas halved and Kmunchanged. 0.99 mol of phosphate was incorporated per tetrameric molecule of D-glyceraldehyde-3-phosphate dehydrogenase under the experimental conditions employed.

Monomeric forms of E. coliglyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting KMvalues close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.

Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble 3-phosphoglycerate kinase. The strength of the association appeared to depend upon the functional state of both enzymes. The holoform of the dehydrogenase exhibited a lower affinity for the kinase than the enzyme-3-phosphoglycerol·NADH complex. The substrate-free 3-phosphoglycerate kinase associated much stronger with the acylated dehydrogenase than the kinase in complex with 1,3-diphosphoglycerate. Electron-microscopic evidence for the association of the soluble acyl-glyceraldehyde-3-phosphate dehydrogenase·NADH complex and 3-phosphoglycerate kinase was also obtained.

Rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble 3‐phosphoglycerate kinase. The strength of the association appeared to depend upon the functional state of both enzymes. The holoform of the dehydrogenase exhibited a lower affinity for the kinase than the enzyme‐3‐phosphoglycerol·NADH complex. The substrate‐free 3‐phosphoglycerate kinase associated much stronger with the acylated dehydrogenase than the kinase in complex with 1,3‐diphosphoglycerate. Electron‐microscopic evidence for the association of the soluble acyl‐glyceraldehyde‐3‐phosphate dehydrogenase·NADH complex and 3‐phosphoglycerate kinase was also obtained.

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.

Rabbit muscle D‐glyceraldehyde‐3‐phosphate dehydrogenase was shown to serve as a substrate for Ca2+/calmodulin‐dependent protein kinase II with a Kmof 0.33 μM and a Vmaxof 2.63 μmol · min−1· mg−1at pH 7.5 and 30°C. In the absence of calmodulin, the Vmaxwas halved and Kmunchanged. 0.99 mol of phosphate was incorporated per tetrameric molecule of D‐glyceraldehyde‐3‐phosphate dehydrogenase under the experimental conditions employed.

Interaction between protein disulfide isomerase, possessing not only isomerase but also chaperone‐like activity, and olygomeric enzyme, GAPDH, has been studied using technique of immobilization on insoluble support. PDI dimers bound to CNBr‐activated Sepharose were shown to posses high TPOR activity as well as the ability to reactivate lysozyme. Immobilized PDI was not found to interact neither with soluble tetrameric GAPDH, nor with soluble denatured GAPDH. However, soluble PDI binds effectively to immobilized GAPDH monomers; Kdwas found to be 3.7×10−6M, stoichiometry 0.824 mole PDI monomers per mole GAPDH monomers. Immobilized GAPDH tetramers do not interact with PDI. These observations are also confirmed by the data on electrophoresis of proteins bound to immobilized GAPDH monomers and tetramers. The ability of PDI to interact with denatured protein form, but not with the native one, is considered to be evidence of chaperone‐like activity of the enzyme.

Interaction of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) and ladate dehydrogenase with human erythrocyte membranes was studied. Under the conditions of low ionic strength, both enzymes bound to the membranes with similar affinities (Kd ≈ 1 μM). The binding was accompanied by complete inhibition of GPDH and by a 65–75% inhibition of ladate dehydrogenase (LDH). Increasing the ionic strength to physiologically meaningful values (0.15M) completely abolished the inactivation of both dehydrogenases in the presence of erythrocyte membranes, but did not preclude their binding. These results suggest that different modes of enzyme-membrane interaction can be realized under the conditions of low and high ionic strength. They also indicate that GPDH and LDH are capable of functioning in a membrane-bound state.

Examination of the properties ofEscherichia coliand rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenases (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction. The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case ofE. colienzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH. These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries. Modification of the arginine produces marked changes in the shape of the charge transfer complex spectrum in the region of 300–370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD+, although the coenzyme binding characteristics remain largely unaltered. On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction.

Incorporation of l‐[35S]cysteine into rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase was detected following incubation of the enzyme in a mixture containing glyceraldehyde‐3‐phosphate, NAD+and the labeled cysteine. Insignificant binding occurred in the absence of either the substrate or NAD+, suggesting that formation of an acylated enzyme form was a prerequisite for the binding. Stoichiometry of the binding depended on the number of functioning active centers; up to 4 moles of l[35S]cysteine bound per mole tetramer with fresh enzyme preparations. The l‐[35S]cysteine incorporation depended on pH and was maximal when a group having pKaof 8.5 is protonated. To clarify the relevance of this finding to the effect of SH‐containing compounds previously shown to decrease the rate of 3‐phosphoglyceroyl‐enzyme hydrolysis [Kuzminskaya et al., FEBS Lett. 336 (1993) 208–210], the pH‐dependence of the effect of glutathione on the hydrolysis rate was determined and found to be close to the pH‐dependence of l‐[35S]cysteine binding.

E. Colid‐glyceraldehyde‐3‐phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli3‐phosphoglycerate kinase with a stoichiometry of 1.77±0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kdof 1.03±0.68μM (10 mM sodium phosphate, 0.15 M NaCl). No interaction was detected between E. colid‐glyceraldehyde‐3‐phosphate dehydrogenase and rabbit muscle 3‐phosphoglycerate kinase. The species‐specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E. colid‐glyceraldehyde‐3‐phosphate dehydrogenase and E. coli3‐phosphoglycerate kinase, which consists of an increase in steady‐state rate of the coupled reaction.

E. Colid-glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli3-phosphoglycerate kinase with a stoichiometry of 1.77±0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kdof 1.03±0.68μM (10 mM sodium phosphate, 0.15 M NaCl). No interaction was detected between E. colid-glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle 3-phosphoglycerate kinase. The species-specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E. colid-glyceraldehyde-3-phosphate dehydrogenase and E. coli3-phosphoglycerate kinase, which consists of an increase in steady-state rate of the coupled reaction.