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.     

The inhibition of glyceraldehyde-3-phosphate dehydrogenase by nitroxyl (HNO)

Nitroxyl (HNO) has received recent and significant interest due to its novel and potentially important pharmacology. However, the chemical/biochemical mechanism(s) responsible for its biological activity remain to be established. Some of the most important biological targets for HNO are thiols and thiol proteins. Consistent with this, it was recently reported that HNO inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein with a catalytically important cysteine thiol at its active site. Interestingly, it was reported that intracellular GAPDH inhibition occurred without significantly altering the cellular thiol redox status of glutathione. Herein, the nature of this reaction specificity was examined. HNO is found to irreversibly inhibit GAPDH in a manner that can be protected against by one of its substrates, glyceraldehyde-3-phosphate (G-3-P). These results are consistent with the idea that HNO has the ability to react with and oxidize a variety of intracellular thiols and the ease or facility of cellular re-reduction of the thiol targets can determine the target specificity.     

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.     

The maize cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family: organ-specific expression and genetic analysis

Summary The distribution of the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family (Gpc) in the maize genome was investigated; a genetic variant of glyceraldehyde-3-phosphate dehydrogenase activity is also described. Restriction fragment length polymorphism analysis of an F₂ population shows that the variant is not linked to the three known Gpc genes. However, this trait is linked to one of two genomic DNA fragments that hybridize to a fragment of the Gpc3 coding region, implying the existence of a fourth Gpc gene. Antibodies and cDNA clones were used to investigate the organ-specific expression of the Gpc genes. Results were compared with the expression of the alcohol dehydrogenase 1 (Adh1) gene. RNA and protein levels were examined in seedling roots and shoots, as well as the leaves, developing endosperm and embryo, and the aleurone. In general, it was found that Gpc3 expression behaves in parallel with Adh1 in these organs, and protein levels closely parallel that of RNA for each gene examined. Both Gpc3 and Adh1 show a marked increase in expression during endosperm development, reaching a maximum 15 days after pollination, but no expression is detected in the leaf. Gpc1 expression is similar to that of Gpc2, with an overall decrease in the level of RNA during endosperm development. This expression is discussed in terms of the common sequences found upstream of genes expressed in the developing maize seed.     

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     

Isolation and properties of an isocitrate dehydrogenase from Anacystis nidulans

A NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) was isolated and purified over 400-fold from Anacystis nidulans. The enzyme activity responded slowly to rapid changes in ligand (NADP⁺, isocitrate, Mg²⁺-ions) or enzyme concentration as well as to rapid changes in temperature. These are properties characteristic of the hysteretic enzymes. In addition, the enzyme activity was subject to product (-ketoglutarate) inhibition. ATP, ADP and CDP also inhibited the enzyme. Unlike several other cyanobacterial enzymes, the isocitrate dehydrogenase of Anacystis is not under redox control.     

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     

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.     

Oxidation of glyceraldehyde-3-phosphate dehydrogenase enhances its binding to nucleic acids

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with various activities far from its enzymatic function. Here, we showed that the oxidation of SH-groups of the active site of GAPDH enhanced its binding with total transfer RNA or with total DNA. Both NAD and NADH-the cofactors of GAPDH-inhibited the GAPDH-RNA (DNA) interaction, though NAD was much less effective than NADH in the case of oxidized GAPDH. Oxidation of GAPDH strongly decreased its affinity to NAD but not to NADH. Immobilized tetramers of GAPDH dissociated into dimers during the incubation with total RNA but not DNA. The staining of HeLa cells with monoclonal antibodies specific to dimers, monomers or the denatured form of GAPDH revealed the condensation of non-native forms of GAPDH in the nucleus. The role of oxidation of GAPDH in the regulation of the quaternary structure of the enzyme and in its interaction with nucleic acids is discussed.     

Acrylonitrile irreversibly inactivates glyceraldehyde-3-phosphate dehydrogenase by alkylating the catalytically active cysteine 149

Acrylonitrile (AN) is a vinyl monomer used in the production of synthetic fibers, rubber and plastics. AN is acutely toxic but its mechanism of toxicity remains to be established. AN is metabolized to cyanide in vivo but cyanide production alone cannot explain acute AN toxicity. Previous work in our laboratory has shown that AN can alkylate highly reactive cysteine residues in proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a critical enzyme involved in glycolysis, has a catalytically active cysteine 149 in its active site. We report that AN irreversibly inhibits GAPDH with second-order rate constants, at pH 7.4, of 3.7 and 9.2 M⁻¹ s⁻¹ at 25 and 37 °C, respectively. A combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and electrospray ionization–mass spectrometry–mass spectrometry (ESI–MS–MS) was used to show that AN inactivates GAPDH by covalently binding to cysteine 149 in the active site of the enzyme. Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and when coupled with the inhibition of mitochondrial ATP synthesis by the AN metabolite cyanide would result in metabolic arrest. The brain can withstand metabolic arrest for only a few minutes thus these combined actions may account for the acute toxicity of AN in vivo.     

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis

Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.     

Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene

Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species.

In the present study, the 931–934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates.

These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.     

Effect of gibberellic acid on photosynthesis and glycollate dehydrogenase in Anacystis nidulans

Gibberellic acid at 10^-4 Mxxx was optimal for enhancement of growth, O₂ evolution, photosystem II and I and the activity of glycollate dehydrogenase of Anacystis nidulans. A stimulatory effect was observed on photosystem II. Other concentrations of gibberellic acid were inhibitory to O₂ evolution and photosystem I. Syntheses of phycocyanin, phycoerythrin and -carotene were significantly enhanced after 48 h incubation with gibberellic acid at 10^-3 Mxxx but the chlorophyll content began to increase 3 h after adding 10^-4 Mxxx gibberellic acid.The author is with the Department of Biological Sciences, Faculty of Science, University of Science and Technology, Irbid, Jordan.     

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.     

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 μM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly.     

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.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase of human malignant cells by methylglyoxal

The effect of methylglyoxal on the activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PD) of several normal human tissues and benign and malignant tumors has been tested. Methylglyoxal inactivated GA3PD of all the malignant cells (47 samples) and the degree of inactivation was in the range of 25-90%, but it had no inhibitory effect on this enzyme from several normal cells (24 samples) and benign tumors (13 samples). When the effect of methylglyoxal on other two dehydrogenases namely glucose 6-phosphate dehydrogenase (G6PD) and L-lactic dehydrogenase (LDH) of similar cells was tested as controls it has been observed that methylglyoxal has some inactivating effect on G6PD of all the normal, benign and malignant samples tested, whereas, LDH remained completely unaffected. These studies indicate that the inactivating effect of methylglyoxal on GA3PD specifically of the malignant cells may be a common feature of all the malignant cells, and this phenomenon can be used as a simple and rapid device for the detection of malignancy.