Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus

The NAD＋-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase （GAPDH; EC 1.2.1.12） was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants （Kin） for NAD＋ and D-glyceraldehyde-3-phosphate were 92.0 μM and 73.4 μM, respectively. The maximal velocity （Vmax） of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 ℃. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform （pI 7.9）.     

Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes

A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase （GAPDH, EC 1.2.1.12） from human erythrocytes. The comparison between this rapid method （one step） and the tra- ditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of -150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide （NAD＋）. Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively. The kinetic parameters were also calculated： the Vmax was 4.3 U/mg and the Km values against G3P and NAD＋ were 20.7 and 17.8 μM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.     

Investigation of glyceraldehyde-3-phosphate dehydrogenase from human sperms

Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS-PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.     

Purification and characterization of cytosolic glycerol-3-phosphate dehydrogenase from skeletal muscle of jerboa (Jaculus orientalis)

Cytosolic glycerol-3-phosphate dehydrogenase was purified from jerboa (Jaculus orientalis) skeletal muscle and its physical and kinetic properties investigated. The purification method consisted of a multi-step procedure and this procedure is presented. The specific activity of the purified enzyme is 53.6 U/mg of protein, representing a 77-fold increase in specific activity. The apparent Michaelis constant (K_m) for dihydroxyacetone is 137.39 (± 25.56) M whereas the K_m for glycerol-3-phosphate is 468.66 (±27.59) M. The kinetic mechanism of purified enzyme is ordered Bi-Bi and this result is confirmed by the product inhibition pattern. Under the conditions of assay, the pH optimum occurs at pH 7.7 for the reduction of dihydroxyacetone phosphate and at pH 9.0 for glycerol-3-phosphate oxidation. In the direction of dihydroxyacetone phosphate, the optimal temperature is 35°C. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 33,000 (±1,000), whereas non-denaturing polyacrylamide gel yields a molecular weight of 72,000 (±2,000), suggesting that the enzyme may exist as a dimer. A polyclonal antiserum raised against the purified enzyme was used to localize the enzyme in different jerboa tissues by Western blot method. The purified enzyme is sensitive to N-ethylmaleimide, and incubation of the enzyme with 20 mm N-ethylmaleimide resulted in a complete loss of catalytic activity. The purified enzyme is inhibited by several metal ions including Zn²⁺ and by 2,4-dichlorophenoxyacetic acid.     

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.     

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS–PAGE yielded a set of bands in the range of 50–60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings.The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.     

Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli

Streptococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60°C with activation energy of 51 KJ mole^–. The apparent K_m values for NADP and D-G3P or DL-G3P were estimated to be 0.385 ± 0.05 and 0.666 ± 0.1 mM, respectively and the V_max of the purified protein was estimated to be 162.5 U mg^–1. The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.     

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the T_m values of the heat-absorption curves decreased by 6.0 and 3.3 °C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD⁺-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35 M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311–H124 are significant for the enhanced stability of GAPDS. The salt bridge D311–H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.     

球毛壳菌甘油醛-3-磷酸脱氢酶基因克隆及特性分析

用粗糙脉孢菌(Neurospora crassa,XP_327967)和菜豆炭疽病菌(Colletotrichum lindemuthianu,P35143)的甘油醛_3_磷酸脱氢酶基因(Glyceraldehyde 3_phosphatedehydrogenase,GAPDH)氨基酸序列对球毛壳菌(Chaetomium globosum)菌丝ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌GAPDH全长cDNA序列。该序列长1240bp,开放阅读框1014bp,编码337个氨基酸组成的多肽,蛋白分子量为36.1kD。用PCR方法克隆了该基因的DNA序列,序列长为1556bp,由2个内含子和3个外显子组成。BlastP同源性分析表明该基因与鹅掌柄孢壳(Podosporaanserine)同源性最高为95%;与米曲霉(Aspergillusoryzae)同源性最低为87%。GAPDH酵母转化子生物功能分析表明转化子对Na2CO3和高温有高的耐受性,证明GAPDH为抗胁迫基因。该基因的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY522719,AY593253,AAS01412)。     

Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus^® cells and the pRSETB vector was successful for expression of a soluble His₆-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.     

Regulation of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by ferriprotoporphyrin IX

Erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme containing critical thiol groups and whose activity is reversibly inhibited by binding to the cell membrane. Here, we demonstrate that the insertion of ferriprotoporphyrin IX (FP) into the red cell membranes exerts two opposite effects on membrane bound G3PD. First, the enzyme is partially inactivated through oxidation of critical thiols. Dithiothreitol restores part of the activity, but some critical thiols are irreversibly oxidized or crosslinked to products of FP-induced lipid peroxidation. Second, G3PD binding to the membrane is modified and the enzyme is activated through displacement into the cytosol and/or release from its binding site.     

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.     

Dietary level of protein regulates glyceraldehyde-3-phosphate dehydrogenase content and synthesis rate in mouse liver cytosol

The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a Mr 36 000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The Mr 36 000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers.     

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.     

Immunocytochemical localization of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in soybean nodules

Polyclonal antibodies raised against NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase [phosphorylating], EC 1.2.1.12) from the plant cytosolic fraction of soybean [ Glycine max (L.) Merr. cv. Williams] nodules were used to study the subcellular location of the enzyme and its relative distribution between infected, interstitial and cortical cells of soybean (cv. Lincoln) nodules. Post-embedding immunogold labelling was carried out on nodules harvested 5, 12, 19 and 25 days after the first sign of nodulation. Labelling for glyceraldehyde-3-phosphate dehydrogenase was observed over the cytoplasm and nuclei of infected and uninfected cells, as well as over the nucleoid regions of bacteroids. In 5-day-old nodules, label also bound adjacent to the peribacteroid membranes. Statistical analysis of the number of gold particles per cell area indicated that in 5-day-old nodules, glyceraldehyde-3-phosphate dehydrogenase was distributed equally between infected, interstitial and cortical cells, but in older nodules the enzyme was more prominent in the interstitial and cortical cells than in infected cells.     

Cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene and the use of its promoter for expression in Myrothecium gramineum, a novel expression host

At our laboratory, research has focused on the development of Myrothecium gramineum as a novel expression host. The glyceraldehyde-3-phosphate dehydrogenase (gpd)-promoter of M. gramineum was isolated and characterized (Genbank accession number EF486690). In order to prove its functionality and to explore the potential of M. gramineum as a novel fungal expression host, use of this gpd-promoter for the expression of a fungal alpha-amylase was investigated. Myrothecium gramineum was transformed with pGPDlpAmyAO, containing the gpd-promoter followed by the amy3 encoding sequence of Aspergillus oryzae. Study of the amylase production indicated that the promoter can be successfully used for the expression of heterologous proteins in M. gramineum. To the best of our knowledge, this is the first time a homologous expression system has been described for M. gramineum.