Interaction between mammalian glyceraldehyde-3-phosphate dehydrogenase and L-lactate dehydrogenase from heart and muscle

The exceptionally high protein concentration in living cells can favor functional protein-protein interactions that can be difficult to detect with purified proteins. In this study we describe specific interactions between mammalian D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) isozymes from heart and muscle. We use poly(ethylene-glycol) (PEG)-induced coprecipitation and native agarose electrophoresis as two independent methods uniquely suited to mimic some of the conditions that can favor protein-protein interaction in living cells. We found that GAPDH interacts with heart or muscle isozymes of LDH with approximately one-to-one stoichiometry. The interaction is specific; GAPDH shows interaction with two LDH isozymes that have very different net charge and solubility in PEG solution, while no interaction is observed with GAPDH from other species, other NAD(H) dehydrogenases, or other proteins that have very similar net charge and molecular mass. Analytical ultracentrifugation showed that the LDH and GAPDH complex is insoluble in PEG solution. The interaction is abolished by saturation with NADH, but not by saturation with NAD(+) in correlation with GAPDH solubility in PEG solution. The crystal structures show that GAPDH and LDH isozymes share complementary size, shape, and electric potential surrounding the active sites. The presented results suggest that GAPDH and LDH have a functional interaction that can affect NAD(+)/NADH metabolism and glycolysis in living cells.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Effect of selected mercurials on glyceraldehyde-3-phosphate-dehydrogenase in Pteronarcys californica

Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was extracted from Pteronarcys californica (Plecoptera) by ammonium sulphate fractionation. The enzyme was treated with three forms of mercury (mercuric chloride, methylmercuric chloride, and phenylmercuric chloride) and their in vitro and in vivo effects on GAPDH were studied. It was found that the orders of toxicity were reversed in the in vivo-in vitro experiments. Electrophoresis was conducted on both treated and untreated enzyme, and showed no differences in mobilities between the two. The enzyme was not found to be inhibited by 0·1 mM iodoacetic acid and required cysteine for activity.     

Interaction of glyceraldehyde-3-phosphate dehydrogenase in the light-induced rod alpha-transducin translocation

The light-dependent subcellular translocation of rod alpha-transducin (GNAT-1, or rod Tα) has been well documented. In dark-adapted animals, rod Tα (rTα) is predominantly located in the rod outer segment (ROS) and translocates into the rod inner segment (RIS) upon exposure to the light. Neither the molecular participants nor the mechanism(s) involved in this protein trafficking are known. We hypothesized that other proteins must interact with rTα to affect the translocations. Using the MBP-rTα fusion pulldown assay, the yeast two-hybrid assay and the co-immunoprecipitation assay, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rTα as interacting proteins. Immunoprecipitation also showed β-actin associates with rTα in the dark but not in the light. To further investigate the involvement of GAPDH in light-induced rod Tα translocation, GAPDH mRNA was knocked down in vivo by transient expression of siRNAs in rat photoreceptor cells. Under completely dark- and light-adapted conditions, the translocation of rTα was not significantly different within the 'GAPDH knock-down photoreceptor cells' compared to the non-transfected control cells. However, under partial dark-adaptation, rTα translocated more slowly in the 'GAPDH knock-down cells' supporting the conclusion that GAPDH is involved in rTα translocation from the RIS to the ROS during dark adaptation.     

Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)

Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300 μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300 μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Studies on the nature of the subcellular localization of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in chicken skeletal muscle

The elucidation of the subcellular localization of enzymes by the classical technique of homogenization followed by differential centrifugation is limited in that it is difficult to determine the effect of the severe disruptive procedures on the normal relationship of the enzymes to their subcellular environment. Attempts have been made to study this problem under less severe limitations; one of the approaches used has been the use of pressure on whole muscle tissue to extract the cellular fluids. In this report we introduce the concept of “comparative extraction” for evaluation of results obtained by this procedure. By comparing the efflux of enzymes of similar solubility and similar size and shape, it is possible to determine the minimal amount of the less easily extractable enzyme which cannot be removed due to compartmentation or binding to cellular particulate structures. Using this concept of “comparative extraction,” we show in this report that at least 35% of the lactate dehydrogenase of chicken breast muscle is restricted in its removal. The data do not definitely resolve the problem of whether the restriction is due to compartmentation of the enzyme within subcellular organelles or binding to subcellular structures.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD(+), and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.     

The Muridae glyceraldehyde-3-phosphate dehydrogenase family

Summary Although only one gene is known to be functional, numerous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences are scattered throughoutMus musculus andRattus rattus genomes. In this report we show that: (1) GAPDH pseudogenes are repeated to comparable extents, at least 400 copies, in 12 other Muridae species; (2) the complete, or nearly so, sequence of GAPDH messenger RNA is amplified, and a high proportion, if not all of these copies, are intronless; (3) GAPDH pseudogenes are preferentially located in heavily methylated and DNAse I-insensitive regions of chromatin; and (4) the presence of atypical GAPDH-related mRNAs in different cellular contexts raises the possibility that more than one GAPDH gene is transcribed.     

Interaction of lactate dehydrogenase with skeletal muscle troponin

Rabbit muscle troponin complex covalently bound to CNBr-activated Sepharose 4B was shown to interact with soluble lactate dehydrogenase with a stoichiometry of 2 mol lactate dehydrogenase/mol of troponin. The presence of Ca2+ influenced the strength of association (the KD values of 0.73 and 2.3 microM were determined in the presence of 200 microM EGTA or 100 microM Ca2+, respectively). In the absence of Ca2+, the affinity of lactate dehydrogenase to troponin was strongly pH-dependent, reaching a maximum in the region of pH 6.0-7.0. No change of catalytic activity was observed as a result of interaction between lactate dehydrogenase and troponin, the enzyme appeared capable of functioning in the bound form.     

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

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The radiolysis of glyceraldehyde-3-phosphate dehydrogenase.

The yields in molecules per 100 eV for active-site and sulphydryl loss from glyceraldehyde-3-phosphate dehydrogenase have been determined in nitrous-oxide-saturated, aerated and argon-saturated solutions. Molecular hydrogen peroxide produces a sulphenic acid product, which can be repaired by post-irradiation treatment with dithiothreitol. Comparison of the yields under various conditions showed that in aerated solutions both .OH and .O2-radicals inactivated the enzyme with an efficiency of about 26 per cent. However, the efficiency of .OH in air-free solutions was less, and inactivation by .H and eaq- did not appear to be appreciable. There is a correlation between SH loss and loss of active sites.