Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP⁺, with K_m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K_m of 0.31 mM. The NADP⁺ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K_m and V_max values with increasing NADP⁺, denoting negative cooperativity. The two K_m's for high and low NADP⁺ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP⁺ binding sites.     

X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Two X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase (G6PD) were found among the progenies of isogenic strains which had been subjected to selection for high G6PD activity. Mapping of the high-activity factor in these mutants was carried out using car Zw^Bsw males of low G6PD activity. As a result, the factor mapped 0.02–0.04 unit to the left of the Zw locus. The amount of the G6PD gene was also quantitated utilizing a cloned G6PD gene as a probe, but no significant difference was found between the mutants and low-G6PD activity flies which shared the same X, second, and third chromosomes with the mutants. These findings are consistent with our notion that the mutations might be regulatory mutations, possibly resulting from the insertion of a novel class of transposable genetic elements.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Characterization of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase from hazel cotyledons

NADP reduction was shown to occur in a crude cytosolic extract from the cotyledonary material of hazel seed prior to the addition of erogenous dehydrogenase substrate. This activity interfered with the assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities. The inherent NADP reduction was removed by ammonium sulphate fractionation. Subsequent de-salting of the resulting partially-purified fraction permitted assay of G6PDH and 6PGDH. Both enzymes were shown to be NADP specific. Typical Michaelis-Menten kinetics were shown for each enzyme, towards NADP and their respective substrate.     

Rabbit bone marrow glucose-6-phosphate dehydrogenase during erythroid cell development

Studies were carried out on glucose-6-phosphate dehydrogenase (G6P-DH) during the differentiation of rabbit bone marrow erythroid cells. It was found that G6P-DH, although displaying a 7-fold activity decrease, did not change the relative amounts of its three dimeric forms.Using homogeneous enzyme preparations, we observed that from dividing to non-dividing erythroblasts the following properties remained constant: V max dependence on pH and temperature, Km for G6P dependence on pH, heat stability, 2-deoxy glucose-6-phosphate utilization, molecular weight, while the Km for NADP significantly increased in non-dividing erythroblasts. These results indicate that no shift towards the oxidized form of the enzyme and no substantial modifications of the protein take place during cell differentiation.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.     

Eukaryotic glucose-6-phosphate dehydrogenases: Structural screening of related proteins

Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described. Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures. Affinity labeling allows further correlations. The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.     

X-linked glucose-6-phosphate dehydrogenase deficiency inMus musculus

A mouse with X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency has been recovered in offspring of 1-ethyl-1-nitrosourea-treated male mice. The activity alteration was detected in blood but can also be observed in other tissue extracts. Hemizygous, heterozygous, and homozygous mutants have, respectively, about 15, 60, and 15% G6PD remaining activity in the blood as compared to the wild type. Erythrocyte indices did not show differences between mutants and wild types. The mutation does not affect the electrophoretic migration, the isoelectric point, or the thermal stability. Kinetic properties, such as theK_m for glucose-6-phosphate or for NADP and the relative utilization of substrate analogues, showed no differences between wild types and mutants with the exception of the relative utilization of deamino-NADP which was significantly lower in mutants. This is presently the only animal model for X-linked G6PD deficiency in humans.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.     

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.     

The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes

A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.     

Cytochemical observations on glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and -glycerophosphate dehydrogenase in chick liver cell cultures infected with Trichomonas vaginalis

Cytochemical reactions specific for glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and α-glycero-phosphate dehydrogenase were observed in the epithelial cells and macrophages of chick liver cell cultures; α-glycerophosphate dehydrogenase activity was observed also in the fibroblasts. Distribution of three of the enzymes was limited to the cytoplasm, their activity being localized primarily in cytoplasmic inclusions. Weak staining of the nuclei and strong staining of the nucleoli occurred in addition to the cytoplasmic reaction in cells treated for glucose-6-phosphatase. In cell cultures inoculated with Trichomonas vaginalis, the activity of three of the enzymes decreased progressively in the course of infection, but that of α-glycerophosphate dehydrogenase increased.     

Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in Penicillium chrysogenum

Abstract The specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase changed when Penicillium chrysogenum was grown on different carbon sources. In the presence of 2% lactose, the activities of these enzymes were approximately 25–35% lower than those in media containing 2% glucose or 2% fructose. We assume that an increase in cAMP concentration was responsible for the observed decreases in the enzyme activities, because a higher cAMP concentration could be detected when the mycelium was grown in a medium containing solely lactose as carbon source. The likely role played by cAMP in the regulation was also demonstrated by the addition of either cAMP or caffeine to the medium.     

The effect of wounding on glucose-6-phosphate dehydrogenase of Solanum tuberosum tuber tissue

Two forms of glucose-6-phosphate dehydrogenase were separated by disc electrophoresis of potato tuber extracts. The slower moving enzyme has a MW of 260 000 the faster one of 130 000. Wounding of potato tubers enhances the relative activity of the slower moving enzyme. Addition of NADP⁺ to the cathode buffer during electrophoresis has the same effect as wounding, whereas addition of glucose-6-phosphate has an opposite effect. The role of the wound induced increase of the pyridine nucleotide level in the interconversion of the two forms of glucose-6-phosphate dehydrogenase is discussed.     

Glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase of wild oat seeds

The presence of the initial enzymes of the pentose phosphate pathway, namely glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase, has been demonstrated in dormant seed of wild oat. Before a partial characterization of these enzymes was made, an inherent NADP-reducing activity and an enzyme deactivating component, both present in the crude extract, were removed by ammonium sulphate precipitation and subsequent desalting. Both enzymes were then shown to be NADP-specific. Typical Michaelis-Menten kinetics were shown by each enzyme towards NADP and their respective substrates. Soluble cytoplasmic dehydrogenase enzymes were present in both embryo and endosperm extracts.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Glucose-6-phosphate dehydrogenase activity and protein turnover in erythroblasts separated by velocity sedimentation at unit gravity and percoll gradient centrifugation

This work was undertaken to improve a separation method for preparation of large amounts of erythroid cells of different age with homogeneous and minimal contamination of myeloid cells. Our method was suitably employed in the study of the decay mechanism of glucose-6-phosphate dehydrogenase (G6PDH) during the erythroid cell maturation.Twenty fractions of erythroid cells at different advancing stages of maturation were prepared by fractionating, at unit gravity, bone marrow cells from anaemic rabbit. The specific activity of the G6PDH was assayed and plotted vs the fraction number and the typical sigmoid curve of the activity decay was drawn. The separated cells were then grouped in three sets of fractions following the three phases of the sigmoid curve and the fractions of each set were combined. From the cytochemical analysis of the three main fractions so obtained, we found a 25–30% myeloid cell contamination in the first fraction, while in the other two fractions the myeloid contamination was 10% or less. For this reason we performed a rapid separation of the first fraction on a discontinuous percoll gradient. By this method, the myeloid cell contamination of the first fraction was levelled down to the other two. The fractions, so obtained, (I, II and III in order of increasing cell maturation) showed a four fold decrease of glucose-6-phosphate dehydrogenase activity expressed both per cell number and on protein base. On the contrary the concentration of the total soluble proteins did not change significantly in the three fractions.The three purified cellular populations were used to provide information on the protein turnover of the erythroid cells during their development. We measured, in intact cells, the rate of synthesis and degradation of total proteins and then, in cell lysates, we determined the rate of degradation of G6PDH, purified from rabbit RBC and radiolabeled by reductive methylation with C¹⁴-formaldehyde. The rates of proteolysis obtained with total proteins and methyl-G6PDH clearly indicate that the proteolytic machinery of the erythroblasts reduces its activity during the cell maturation.     

Insulin-like effects of vanadate and selenate on the expression of glucose-6-phosphate dehydrogenase and fatty acid synthase in diabetic rats

Isulin is capable of regulating cellular and metabolic processes as well as gene expression. In recent years, enthusiasm has surfaced for using insulin mimetics to study the mechanism of action of insulin. Vanadata and selenate are two compounds that have been found to mimic the action of insulin on control to blood glucose levels in vivo. Vanadata has also been shown to regulate the expression of several enzymes both in vivo, however, studies concerning selenate's ability to regulate expression have not been reported. In his study we show that administration of vanadate or selenate to streptozotocin-induced diabetic rats not only normalizes blood glucose levels similarly to insulin but also positively affects the expression of two key metabolic enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS). Both G6PDH and FAS activity are significantly decreased in diabetic animals compared to non-diabetic control. Treatment of the diabetic animals with either insulin, vanadate or selenate restored both activities to about 80–90% of control. All treatment conditions exhibited activities significantly higher than those determined for the diabetic group but did not differ significantly from each other. Increases in GPDH or FAS activity are due to increases in mRNA level. Increase in both G6PDH and FAS mRNA was comparable to the observed increase in activity suggesting that regulation of expression by the mimetics occurs pretranslationally.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

Aurinetricarboxylic acid: a potent inhibitor of glucose-6-phosphate dehydrogenase.

Inhibition by aurinetricarboxylic acid (ATA) of glucose-6-phosphate (G6P) dehydrogenase was "competitive" with respect to G6P and "mixed type" with respect to NADP+. Inhibited enzyme bound two molecules of ATA. Kinetic constants, Km, Ki at varying pH suggested possible binding of the inhibitor by the sulfhydryl of the enzyme; of the several enzymes tested only milk xanthine oxidase and G6P dehydrogenase from bovine adrenal was inhibited by ATA.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle