Glyceraldehyde-3-phosphate dehydrogenase is involved in the pathogenesis of Alzheimer's disease.

One of important characteristics of Alzheimer's disease is a persistent oxidative/nitrosative stress caused by pro-oxidant properties of amyloid-beta peptide (Aß) and chronic inflammation in the brain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is easily oxidized under oxidative stress. Numerous data indicate that oxidative modifications of GAPDH in vitro and in cell cultures stimulate GAPDH denaturation and aggregation, and the catalytic cysteine residue Cys152 is important for these processes. Both intracellular and extracellular GAPDH aggregates are toxic for the cells. Interaction of denatured GAPDH with soluble Aß results in mixed insoluble aggregates with increased toxicity. The above-described properties of GAPDH (sensitivity to oxidation and propensity to form aggregates, including mixed aggregates with Aß) determine its role in the pathogenesis of Alzheimer's disease.

S-nitrosylation and S-glutathionylation of GAPDH: Similarities, differences, and relationships.

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation. Incubation of HEK 293 T cells in the presence of H2O2 or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H2O2 or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.

Mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in the presence of methylglyoxal.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be one of the targets of methylglyoxal (MGO), a metabolite of glycolysis that increased in diabetes. However, the mechanism of GAPDH inactivation in the presence of MGO is unclear. The purpose of the work was to study the reaction of GAPDH with MGO and to identify the products of the reaction. It was shown that incubation of recombinant human GAPDH with MGO leads to irreversible inactivation of the enzyme, which is accompanied by a decrease in SH-group content by approximately 3.3 per tetramer GAPDH. MALDI-TOF MS analysis showed that the modification of GAPDH with MGO results in the oxidation of the catalytic cysteine residues (Cys152) to form cysteine-sulfinic acid. In addition, 2 arginine residues (R80 and R234) were identified that react with MGO to form hydroimidazolones. Incubation of SH-SY5Y neuroblastoma cells with MGO resulted in the inactivation of GAPDH and inhibition of glycolysis. The mechanism of GAPDH oxidation in the presence of MGO suggests the participation of superoxide anion, which is formed during the reaction of amino groups with methylglyoxal. The role of GAPDH in protection against the damaging effect of ROS in cells in the case of inefficiency of MGO removal by the GSH-dependent glyoxalase system is discussed.

Products of S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase: Relation between S-nitrosylation and oxidation.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the major targets of NO in cells, especially in neurodegenerative diseases. S-Nitrosylation of GAPDH is accompanied by its translocation into the nucleus with subsequent apoptosis. The product of GAPDH modification by NO is considered to be S-nitrosylated GAPDH (GAPDH-SNO). However, this has not been confirmed by direct methods. METHODS: Products of GAPDH modification in the presence of the NO donor diethylamine NONOate were analyzed by MALDI- and ESI- mass spectrometry methods. RESULTS: The adduct between GAPDH and dimedone was detected by MALDI-MS analysis after incubation of S-nitrosylated GAPDH with dimedone, which points to the formation of cysteine-sulfenic acid (GAPDH-SOH) in the protein. Analysis of the protein hydrolysate revealed the incorporation of dimedone into the catalytic residue Cys150. An additional peak that corresponded to GAPDH-SNO was detected by ESI-MS analysis in GAPDH after the incubation with the NO donor. The content of GAPDH-SNO and GAPDH-SOH in the modified GAPDH was evaluated by different approaches and constituted 2.3 and 0.7 mol per mol GAPDH, respectively. A small fraction of GAPDH was irreversibly inactivated after NO treatment, suggesting that a minor part of the products includes cysteine-sulfinic or cysteine-sulfonic acids. CONCLUSIONS: The main products of GAPDH modification by NO are GAPDH-SNO and GAPDH-SOH that is presumably formed due to the hydrolysis of GAPDH-SNO. GENERAL SIGNIFICANCE: The obtained results are important for understanding the molecular mechanism of redox regulation of cell functions and the role of GAPDH in the development of neurodegenerative disorders.

[Expression of sperm-specific glyceraldehyde-3-phosphate dehydrogenase in melanoma cells changes their energy metabolism]. / Ékspressiia spermospetsifichnoi glitseral'degid-3-fosfatdegidrogenazy v melanomnykh kletkakh izmeniaet ikh énergeticheskii obmen.

The somatic isoform of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC1.2.1.12) is involved in such crucial for cancer cells development pathways as induction of apoptosis and glycolytic regulation. At the same time, sperm-specific isoform (GAPDHS) does not exhibit all the same functions as somatic enzyme. The expression of sperm-specific GAPDH without N-terminal domain in some melanoma cells along with somatic isoenzyme, shown in our previous work, has led to the proposal of this unusual enzyme's possible role in regulation of cancer cells glycolysis. In the presented work we have tested production of GAPDHS in 13 additional melanoma cell lines by immunoblotting. We have also gathered data on energy metabolism in 5 selected cell lines by evaluation of glucose uptake and lactate production in differing conditions. We have demonstrated that in standard cultivation media glucose uptake by MelP cells, producing substantial amounts of GAPDHS protein was higher than in MelKor cells, producing lesser amounts of GAPDHS. All other analyzed cell lines that do not produce GAPDHS (MelMS, MelSi and Malme3M) had even a lower glucose uptake rate.

α-Synuclein Overexpression in SH-SY5Y Human Neuroblastoma Cells Leads to the Accumulation of Thioflavin S-positive Aggregates and Impairment of Glycolysis.

Deterioration of energy metabolism in affected cells is an important feature of synucleinopathies, including Parkinson's disease. Here, we studied the association between α-synuclein accumulation and glycolysis using SH-SY5Y neuroblastoma cell lines stably expressing wild-type α-synuclein or its A53T mutant linked to the autosomal dominant form of the disease. Overexpression of both proteins led to the accumulation of thioflavin S-positive aggregates, more pronounced for α-synuclein A53T. It also caused changes in the cell energy metabolism manifested as a decrease in the lactate accumulation and glucose uptake. Impairments in glycolysis were also accompanied by a decrease in the activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In vitro experiments with purified proteins indicated that GAPDH inactivation might be caused by its binding to the monomeric and oligomeric forms of α-synuclein. Therefore, a decrease in the GAPDH activity induced by its interaction with α-synuclein, might be one of the causes of glucose metabolism deterioration in synucleinopathies.

The chaperonin TRiC is blocked by native and glycated prion protein.

Eukaryotic double-ring chaperonin TRiC is an ATP-dependent protein-folding machine. Most of its substrates are known to form large ordered structures from multiple polypeptide chains. Since these structures are similar to fibrillar and oligomeric forms of amyloidogenic proteins, we hypothesized that TRiC may play a role in the development of neurodegenerative diseases of amyloid nature including prion diseases. Enzyme-linked immunosorbent assay showed that monomeric, oligomeric and fibrillar forms of prion protein (PrP) bind strongly to chaperonin TRiC, whereas glycation reduces the prion protein affinity for chaperonin. Nevertheless, dynamic light scattering, electron microscopy and thioflavin T fluorescence confirmed that all studied forms of PrP undergo an amyloid transformation after interaction with chaperonin, but different forms of prion protein are capable of having different effects on the functional state of TRiC. For example, prion protein monomers completely block its ability to reactivate the chaperonin's natural substrate - sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). At the same time, PrP oligomers and fibrils only partially prevent the reactivation of GAPDS upon the action of TRiC. The monomeric forms of prion protein glycated by methylglyoxal do not inhibit, but only slow down the chaperone-dependent reactivation of GAPDS. Thus, the interaction of amyloidogenic proteins with chaperonins could cause cell malfunction.

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein.

BACKGROUND: We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H2O2 and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated. METHODS: Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH. RESULTS: S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S. CONCLUSIONS: S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1. GENERAL SIGNIFICANCE: The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

Inhibitors of Glyceraldehyde 3-Phosphate Dehydrogenase and Unexpected Effects of Its Reduced Activity.

The review describes the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitors to study the enzyme and to suppress its activity in various cell types. The main problem of selective GAPDH inhibition is a highly conserved nature of the enzyme active site and, especially, Cys150 environment important for the catalytic action of cysteine sulfhydryl group. Numerous attempts to find specific inhibitors of sperm GAPDH and enzymes from Trypanosoma sp. and Mycobacterium tuberculosis that would not inhibit GAPDH of somatic mammalian cells have failed, which has pushed researchers to search for new ways to solve this problem. The sections of the review are devoted to the studies of GAPDH inactivation by reactive oxygen species, glutathione, and glycating agents. The final section discusses possible effects of GAPDH inhibition and inactivation on glycolysis and related metabolic pathways (pentose phosphate pathway, uncoupling of the glycolytic oxidation and phosphorylation, etc.).

Polymyxins interaction to the human serum albumin: A thermodynamic and computational study.

Polymyxin B and E (colistin), are a group of cationic charged cyclic antibiotic lipopeptides that are frequently used in the clinics to treat infections caused by the multidrug-resistant gram-negative bacteria. Since the interactions with the blood plasma drug-transport proteins may play a critical role in determining their pharmacological and pharmacokinetic profiles, we studied the binding properties of polymyxins to the human serum albumin (HSA) under simulated physiological conditions by the combination of biophysical approaches, such as isothermal titration calorimetry (ITC), fluorescence anisotropy, circular dichroism (CD) buttressed by computational studies. The HSA binding to the polymyxins was relatively strong (Ka ≈ 1.0 × 107 M-1). Molecular docking indicated that polymyxins bind to the cleft of HSA between domains I and III via the electrostatic interactions. This evidence was further confirmed by the entropy-driven interaction for the polymyxins bound HSA. Far UV-CD experiments showed that the secondary structure of HSA doesn't alter and its stable structure is preserved. Collectively, these investigations revealed that the polymyxins bind preferentially to the partially unfolded intermediate forms of the protein structure; however, HSA molecule does not undergo any significant conformational changes upon binding. This is promising as it may limit the unfavorable side effects of the medicine. On the whole, the results provide quantitative and qualitative insight of the binding interaction between HSA and polymyxins, which is important in understanding their effect as therapeutic agents.

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.

[A rational approach to obtaining high-specific polyclonal antibodies against recombinant alpha-synuclein]. / Ratsional'nyi podkhod k polucheniiu vysokospetsifichnykh poliklonal'nykh antitel protiv rekombinantnogo al'fa-sinukleina.

The approach for the quick and efficient production ofpolyclonal antibodies tothe target antigen alpha-synuclein has been proposed. Two methods have been employed to purify specific rabbit polyclonal antibodies against recombinant human alpha-synuclein, produced by subcutaneous immunization with complete Freund's adjuvant. It was shown that purification on CNBr-activated Sepharose with immobilized alpha-synuclein resulted in antibody preparation with rabbit serum histidine-rich glycoprotein as a contaminant. Two-stage antibody purification procedure first on Sepharose with immobilized protein G, and then on alpha-synuclein immobilized column helps to avoid contamination and to obtain homogenous antibody preparation. Antibodies recognize different conformations of alpha-synuclein and can be used in a variety of immunochemical approaches, including immunocytochemistry.

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. METHODS: Rabbit muscle GAPDH was S-glutathionylated in the presence of H2O2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. RESULTS: Incubation of GAPDH in the presence of H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD+ and thermal stability. CONCLUSIONS: The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. GENERAL SIGNIFICANCE: The manuscript considers one of the possible mechanisms of redox regulation of cell functions.

Glycation, Glycolysis, and Neurodegenerative Diseases: Is There Any Connection?

This review considers the interrelation between different types of protein glycation, glycolysis, and the development of amyloid neurodegenerative diseases. The primary focus is on the role of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase in changing the concentration of carbonyl compounds - first and foremost, glyceraldehyde-3-phosphate and methylglyoxal. It has been suggested that various modifications of the enzyme - from the oxidation of the sulfhydryl groups of the active site to glycation with sugars - can lead to its inactivation, which causes a direct increase in glyceraldehyde-3-phosphate concentration and an indirect increase in the content of other aldehydes. This "primary inactivation" of glyceraldehyde-3-phosphate dehydrogenase promotes its glycation with aldehydes, including its own substrate, and a further irreversible decrease in its activity. Such a cycle can lead to numerous consequences - from the induction of apoptosis, which is activated by modified forms of the enzyme, to glycation of amyloidogenic proteins by glycolytic aldehydes. Of particular importance during the inhibition of glyceraldehyde-3-phosphate dehydrogenase is an increase in the content of the glycating compound methylglyoxal, which is much more active than reducing sugars (glucose, fructose, and others). In addition, methylglyoxal is formed by two pathways - in the cascade of reactions during glycation and from glycolytic aldehydes. The ability of methylglyoxal to glycate proteins makes it the main participant in this protein modification. We consider the effect of glycation on the pathological transformation of amyloidogenic proteins and peptides - ß-amyloid peptide, α-synuclein, and prions. Our primary focus is on the glycation of monomeric forms of these proteins with methylglyoxal, although most works are dedicated to the analysis of the presence of "advanced glycation end products" in the already formed aggregates and fibrils of amyloid proteins. In our opinion, the modification of aggregates and fibrils is secondary in nature and does not play an important role in the development of neurodegenerative diseases. The glycation of amyloid proteins with carbonyl compounds can be one of the triggers of their transformation into toxic forms. The possible role of glycation of amyloidogenic proteins in the prevention of their modification by ubiquitin and the SUMO proteins due to a disruption of their degradation is separately considered.

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.

Dimerization of Tyr136Cys alpha-synuclein prevents amyloid transformation of wild type alpha-synuclein.

Expression of human alpha-synuclein in E. coli cells is known to result in a mixture of the wild type alpha-synuclein and the protein containing Tyr136Cys substitution due to the translational error. The amount of Cys136 alpha-synuclein (Cys136-AS) may reach approximately 50% of the recombinant protein. The wild-type and Cys136-containing fractions of alpha-synuclein were separated using thiol-Sepharose, and their properties were investigated. In the absence of reducing agents, Cys136-AS forms dimers due to the disulfide bonding. Both wild-type and Cys136 alpha-synuclein preparations are prone to aggregate during prolonged incubation under shaking at pH 4 and 37°C, but only the wild-type alpha-synuclein produces amyloid aggregates. The aggregates produced by either monomeric or dimeric Cys136-AS do not exhibit amyloid properties according to the test with Thioflavin T. Moreover, an admixture of dimeric Cys136-AS prevents the amyloid transformation of the wild-type alpha-synuclein. CD spectroscopy analysis revealed an enhanced content of alpha-helical structures in the aggregates produced by dimeric Cys136-AS. The admixture of Cys136-AS in preparations of human recombinant alpha-synuclein can be a source of erroneous interpretation of experiments on amyloid transformation of this protein.

Denaturing action of adjuvant affects specificity of polyclonal antibodies.

Influence of the immunization procedure on the specificity of the produced antibodies towards different conformations of the antigen was investigated. It was demonstrated that intravenous immunization of a rabbit with an adjuvant-free solution of recombinant sperm-specific glyceraldehyde-3-phosphate dehydrogenase (dN-GAPDS) resulted in production of antibodies recognizing only native conformation of dN-GAPDS and exhibiting no cross-reaction with somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. A subcutaneous immunization with human dN-GAPDS mixed with Freund's complete adjuvant yielded antibodies recognizing both native and denatured conformation of dN-GAPDS. The oil component of the adjuvant was shown to cause inactivation and partial denaturation of dN-GAPDS, leading to exposure of the epitopes that are masked in the native protein, which resulted in production of the antibodies to the denatured antigen. These results may be of importance for biochemical research that often require polyclonal antibodies recognizing different conformations of antigens.

Inhibition of Chaperonin GroEL by a Monomer of Ovine Prion Protein and Its Oligomeric Forms.

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.

Low Concentrations of Hydrogen Peroxide Activate the Antioxidant Defense System in Human Sperm Cells.

The effect of low concentrations of hydrogen peroxide (10-100 µM) on sperm motility and on the activity of the sperm enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) was investigated. Incubation of semen samples with 10 and 100 µM hydrogen peroxide increased the content of spermatozoa with progressive motility by 20 and 18%, respectively, and enhanced the activity of GAPDS in the sperm cells by 27 and 20% compared to a semen sample incubated without additions. It was also found that incubation with 10 µM hydrogen peroxide increased the content of reduced glutathione (GSH) in sperm cells by 50% on average compared to that in the control samples. It is supposed that low concentrations of hydrogen peroxide activate the pentose phosphate pathway, resulting in NADPH synthesis and the reduction of the oxidized glutathione by glutathione reductase yielding GSH. The formed GSH reduces the oxidized cysteine residues of the GAPDS active site, increasing the activity of the enzyme, which in turn enhances the content of sperm cells with progressive motility. Thus, the increase in motile spermatozoa in the presence of low concentrations of hydrogen peroxide can serve as an indicator of normal functioning of the antioxidant defense system in sperm cells.

Structural basis for the NAD binding cooperativity and catalytic characteristics of sperm-specific glyceraldehyde-3-phosphate dehydrogenase.

Catalytic properties of enzymes used in biotechnology can be improved by eliminating those regulatory mechanisms that are not absolutely required for their functioning. We exploited mammalian glyceraldehyde-3-phosphate dehydrogenase as a model protein and examined the structural basis of the NAD(+) cooperative binding exhibited by its homologous isoenzymes: the somatic enzyme (GAPD) and the recombinant sperm-specific enzyme (dN-GAPDS). Moreover, we obtained a mutant dN-GAPDS, which misses the cooperativity, but exhibits a twofold increase in the specific activity instead (92 and 45 µmol NADH/min per mg protein for the mutant and the wild type proteins, respectively). Such an effect was caused by the disruption of the interdomain salt bridge D311-H124, which is located close to the active site of the enzyme. The thermal stability of the mutant protein also increased compared to the wild type form (heat absorption peak values were 70.4 and 68.6 °C, respectively). We expect our findings to be of importance for the purposes of biotechnological applications.