Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol     

Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase inArabidopsis thaliana

Summary NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent GAPDH) was purified to homogeneity and injected into a rabbit to induce a polyclonal antibody. The antibody was judged to be of high specificity and high affinity. This antibody was used to probe sections ofArabidopsis leaf, stem or roots which were fixed using either paraformaldehyde or a high-pressure freezing method. Our results show that the NAD-dependent GAPDH localizes in the nucleus as well as in the cytosol. In phloem tissue, the NAD-dependent GAPDH was found in companion cells but not in the sieve element.     

A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote,Trichomonas vaginalis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD⁺ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica—Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium. Correspondence to: M. Müller     

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: An evolutionary scenario of subcellular compartmentation in Kinetoplastida

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72–77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD⁺-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosome, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) - HK hexokinase (EC 2.7.1.1) - PGI glucosephosphate isomerase (EC 5.3.1.9) - PGK phosphoglycerate kinase (EC 2.7.2.3) - PYK pyruvate kinase (EC 2.7.1.40) - TIM triosephosphate isomerase (EC 5.3.1.1) - SDS sodium dodecyl sulfate - SSC saline sodium citrate (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) - MYR millions of years Nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases under accession number X74535 Correspondence to: P.A.M. Michels     

Decrease of dehydrogenase activity of cerebral glyceraldehyde-3-phosphate dehydrogenase in different animal models of Alzheimer's disease

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the β-amyloid precursor protein (βAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the different animal models of AD: transgenic mice (Tg2576) and rats treated with β-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon γ (INFγ). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of β-amyloid, or thiorphan, an inhibitor of neprilysin involved in β-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFNγ, which can influence the progression of the AD, significantly reduced the enzyme activity.     

Pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase fromStreptomyces arenae is closely related to GAPDH from thermostable eubacteria and plant chloroplasts

Streptomyces arenae produces the antibiotic pentalenolactone, a highly specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During the phase of pentalenolactone production,S. arenae expresses a pentalenolactone-insensitive GAPDH isoform; otherwise, a pentalenolactone-sensitive form is expressed. The gene of the pentalenolactone-insensitive GAPDH was cloned and sequenced. Regulatory elements typical for genes encoding antibiotic resistance and production are localized upstream and downstream of the open reading frame. No expression of pentalenolactone-insensitive GAPDH was detected inStreptomyces lividans transformed with the gene. InEscherichia coli, the gene was expressed from an inducedlac promoter. Amino-terminal sequencing of the heterologously expressed GAPDH proved its identity with pentalenolactone-insensitive GAPDH fromS. arenae. Sequence comparisons with GAPDH from other organisms showed a close relationship to GAPDH of plant chloroplasts, of other gram-positive bacteria, and of thermophilic gram-negative bacteria. Pentalenolactone-insensitive GAPDH differs from all closely related GAPDHs only in a few residues, none of which are directly involved in catalysis or substrate binding. The total amino acid composition is more similar to GAPDH of thermophilic species than to that of mesophilic species. The purified enzyme was moderately thermotolerant, which could be a side effect of the structural changes causing pentalenolactone-resistance.Abbreviations GAP Glyceraldehyde-3-phosphate - GAPDH Glyceraldehyde-3-phosphate dehydrogenase     

Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter

The glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of the sophorolipid producing yeast Candida bombicola was isolated using degenerated PCR and genome walking. The obtained 3,740 bp contain the 1,008 bases of the coding sequence and 1,613 and 783 bp of the upstream and downstream regions, respectively. The corresponding protein shows high homology to the other known GPD genes and is 74% identical to the gyceraldehyde-3-phosphate dehydrogenase of Yarrowia lipolytica. The particular interest in the C. bombicola GPD gene sequence originates from the potential use of its promoter for high and constitutive expression of homologous and heterologous genes. Southern blot analysis did not give any indication for the presence of multiple GPD genes and it can therefore be expected that the promoter can be used for efficient and high expression. This hypothesis was further confirmed by the biased codon usage in the GPD gene. GDP promoter fragments of different lengths were used to construct hygromycin resistance cassettes. The constructs were used for the transformation of C. bombicola and all of them, even the ones with only 190 bp of the GPD promoter, were able to render the cells resistant to hygromycin. The efficacy of a short GPD promoter can be a convenient characteristic for the construction of compact expression cassettes or vectors for C. bombicola. The GenBank accession number of the sequence described in this article is EU315245.     

Enzyme-enzyme interaction in the chloroplast: Glyceraldehyde-3-phosphate dehydrogenase,triose phosphate isomerase and aldolase

Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate and triose-phosphate isomerase bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO₂ fixation and during glycolysis in the chloroplast.Abbreviations FITC fluorescein isothiocyanate - glyceraldehyde3-P glyceraldehyde-3-phosphate - K partition coefficient - K _m (ALD) apparent K _m value obtained when aldolase levels are varied - K _m (GAP) K _m value obtained when glyceraldehyde-3-P concentrations are varied - K _m (PGK) apparent K _m value obtained when phosphoglycerate kinase levels are varied - K _m (TPI) apparent K _m value obtained when triose-P isomerase levels are varied - PEG polyethyleneglycol - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - triose-P triose phosphate We thank Fred J. Stevens, Argonne National Laboratory, for help in analysis of the tertiary structures, Göte Johansson, University of Lund, for hosting two of us in his laboratory where we did the initial phase-partitioning experiments, Chang-hou Li, Shanghai Research Centre of Biotechnology, for the use of the fluorimeter, Lawrence Sykora and the University of Illinois greenhouse staff for growing the pea plants, Jack T. Gibbons for electron microscopy, and Christie Aljets, Xua Ming Da, Xiang He, Arif Ali Khan, Fang Luo, Martha Pacold, Michael Pacold, Lei Shi, Hyun Moon Shin and Qi Zhao for their assistance with these experiments. Support came from the University of Illinois-Chicago Research Board, the US National Science Foundation (Grants DCB 9018265, INT 91-15490 and INT 91-13311) and the Chinese National Science Foundation (Grant 39230050).     

Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: Strategies of protein stabilization

Abstract: The molecular origin of protein stability has been the subject of active research for more than a generation (R. Jaenicke (1991) Eur. J. Biochem. 202, 715–728). Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential. In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima , it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds ( ΔΔG _stab≈ 50 kJ/mol). As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology. In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents.     

Adjustment of conformational flexibility of glyceraldehyde-3-phosphate dehydrogenase as a means of thermal adaptation and allosteric regulation

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Thermotoga maritima (TmGAPDH) is a thermostable enzyme (T (m) = 102 degrees C), which is fully active at temperatures near 80 degrees C but has very low activity at room temperature. In search for an explanation of this behavior, we measured the conformational flexibility of the protein by hydrogen-deuterium exchange and compared the results with those obtained with GAPDH from rabbit muscle (RmGAPDH). At room temperature, the conformational flexibility of TmGAPDH is much less than that of RmGAPDH, but increases with increasing temperature and becomes comparable to that of RmGAPDH near the physiological temperature of Thermotoga maritima. Using the available three-dimensional structures of the two enzymes, we compared the B factors that reflect the local mobility of protein atoms. The largest differences in B factors are seen in the coenzyme and NAD binding regions. The likely reason for the low activity of TmGAPDH at room temperature is that the motions required for enzyme functions are restricted. The findings support the idea of "corresponding states" which claims that over the time span of evolution, the overall conformational flexibility of proteins has been preserved at their corresponding physiological temperatures.     

Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

The white-rot basidiomycete Coriolus versicolor secretes several enzymes that participate in the degradation of lignin and various persistent organic pollutants. In this study, we attempted to establish a genetic transformation system with a homogenous promoter sequence for driving the gene for antibiotic resistance. We succeeded in cloning the promoter sequence of the gene for glyceraldehyde-3-phosphate dehydrogenase (gpd), which is expressed at high levels in C. versicolor. The expression vector pT7GPTHPT was constructed, which included a gene for resistance to hygromycin B under control of the gpd promoter. The successful selection of transformants on medium that contained hygromycin B indicated that the system should be useful not only for the genetic transformation of C. versicolor, but also for the overproduction of useful fungal enzymes such as laccase and peroxidase.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle

Here we describe the interaction of phosphorylated ∼40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Structured summary

MINT-7891324, MINT-7891304, MINT-7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006)     

Glyceraldehyde-3-phosphate dehydrogenase as a biochemical marker of cytotoxicity by vinyl sulfones in cultured murine spleen lymphocytes

Recently, vinyl sulfones have been observed to selectively inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an important ATP-generating enzyme in glycolysis. The possibility of using GAPDH as a biochemical parameter of cytotoxicity by vinyl sulfones was investigated using mouse lymphocytes. Incubation of lymphocyte GAPDH with ethylvinyl sulfone resulted in a pseudo-first-order loss of enzyme activity. The exposure of lymphocytes to ethylvinyl sulfone resulted in the decrease of GAPDH activity followed by ATP depletion and cell death, which were both dependent on the concentration of ethylvinyl sulfone. A further study on the time-dependent change indicated that cell death was preceded by ATP loss. Compared to ethylvinyl sulfone, divinyl sulfone was more than 8 times more potent in causing either ATP depletion or cell death.Abbreviations DTT dithiothreitol - GAPDH glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide     

Starvation and temperature upshift cause an increase in the enzymatically active cell wall-associated glyceraldehyde-3-phosphate dehydrogenase protein in yeast

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity in Saccharomyces cerevisiae increases (two- to 10-fold, depending on the strain) in response to starvation and temperature upshift. Assays using transformants carrying pTDH, a yeast centromer derivative plasmid containing the Candida albicans TDH3 gene (encoding GAPDH) fused in frame with the yeast SUC2-coding region for internal invertase, showed that starvation and/or temperature upshift result in a similar increase in both cwGAPDH and cell wall-associated invertase activities. In addition, this incorporation of GAPDH protein into the cell wall in response to stress does not require (i) de novo protein synthesis, indicating that preexisting cytosolic enzyme is incorporated into the cell wall, (ii) nor the participation of the ubiquitin yeast stress response system, as no differences were observed between wild-type and polyubiquitin-depleted (Deltaubi4) strains.     

Structural analysis of glyceraldehyde-3-phosphate dehydrogenase functional diversity

Multifunctional proteins provide a new mechanism to expand exponentially cell information and capability beyond that indicated by conventional gene analyses. As such, examination of their structure–function relationships provides a means to define the mechanisms through which cells accomplish critical yet disparate activities required for cell viability and survival. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be considered the quintessential multidimensional protein which exhibits a variety of functions unrelated to its classical role in energy production. This review discusses new insights into the structure–function mechanisms through which defined GAPDH amino acid domains are utilized for its diverse activities, the importance of its post-translational modification, and, intriguingly, the logic inherent in the presence or the absence of specific signaling domains.     

Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from the extremely halophilic archaebacterium Haloarcula vallismortis has been purified in a four step procedure to electrophoretic homogeneity. The enzyme is a tetramer with a relative molecular mass of 160000. It is strictly NAD⁺-dependent and exhibits its highest activity in 2 mol/l KCl at 45°C. Amino acid analysis and isoelectric focusing indicate an excess of acidic amino acids. Two parts of the primary sequence are reported. These peptides have been compared with glyceraldehyde 3-phosphate dehydrogenases from other archaebacteria, eubacteria and eucaryotes. The peptides show a high grade of similarity to glyceraldehyde 3-phosphate dehydrogenase from eucaryotes.Abbreviations BCA bicinchoninic acid - CTAB cetyltrimethyl ammonium bromide - DTE dithioerythritol - DTT dithiothreitol - GAP glyccraldehyde 3-phosphate - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Sequence and evolution of the Drosophila pseudoobscura glycerol-3-phosphate dehydrogenase locus

The Gpdh genomic region has been cloned and sequenced in Drosophila pseudoobscura. A total of 6.8 kb of sequence was obtained, encompassing all eight exons of the gene. The exons have been aligned with the sequence from D. melanogaster, and the rates of synonymous and nonsynonymous substitution have been compared to those of other genes sequenced in these two species. Gpdh has the lowest rate of nonsynonymous substitution yet seen in genes sequenced in both D. pseudoobscura and D. melanogaster. No insertion/deletion events were observed, and the overall architecture of the gene (i.e., intron sites, etc.) is conserved. An interesting amino acid reversal was noted between the D. melanogaster Fast allele and the D. pseudoobscura gene.     

Effect of the sodium/potassium ratio on glyceraldehyde-3-phosphate dehydrogenase interaction with red cell vesicles

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the ³¹P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K. (1977) Biochim. Biophys. Acta 464, 82–92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation change induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na⁺ + K⁺)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew ((1970) J. Physiol. London 207, 393–402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by ³¹P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na⁺ + K⁺)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.