NADP+ and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation.

A mild oxidative stimulation of the hexose monophosphate pathway of human glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-deficient erythrocytes (Mediterranean variant) causes a significant drop in NADPH. These results, other than to confirm that glucose-6-phosphate dehydrogenase deficiency is a product deficiency disorder, demonstrate that under oxidative stimulation glutathione reductase may become functionally impaired and GSSG cannot be reduced at a sufficient rate.     

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. Energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes

The effects of various forms of glucose-6-phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism. The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose-6-phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed. It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose-6-phosphate dehydrogenase deficiency.     

Phenylpyruvic Acid decreases glucose-6-phosphate dehydrogenase activity in rat brain

Phenylketonuria is a recessive autosomal disorder that is caused by a deficiency in the activity of phenylalanine-4-hydroxylase, which converts phenylalanine to tyrosine, leading to the accumulation of phenylalanine and its metabolites phenyllactic acid, phenylacetic acid, and phenylpyruvic acid in the blood and tissues of patients. Phenylketonuria is characterized by severe neurological symptoms, but the mechanisms underlying brain damage have not been clarified. Recent studies have shown the involvement of oxidative stress in the neuropathology of hyperphenylalaninemia. Glucose-6-phosphate dehydrogenase plays an important role in antioxidant defense because it is the main source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), providing a reducing power that is essential in protecting cells against oxidative stress. Therefore, the present study investigated the in vitro effect of phenylalanine (0.5, 1, 2.5, and 5?mM) and its metabolites phenyllactic acid, phenylacetic acid, and phenylpyruvic acid (0.2, 0.6, and 1.2?mM) on the activity of enzymes of the pentose phosphate pathway, which is involved in the oxidative phase in rat brain homogenates. 6-Phosphogluconate dehydrogenase activity was not altered by any of the substances tested. Phenylalanine, phenyllactic acid, and phenylacetic acid had no effect on glucose-6-phosphate dehydrogenase activity. Phenylpyruvic acid significantly reduced glucose-6-phosphate dehydrogenase activity without pre-incubation and after 1?h of pre-incubation with the homogenates. The inhibition of glucose-6-phosphate dehydrogenase activity caused by phenylpyruvic acid could elicit an impairment of NADPH production and might eventually alter the cellular redox status. The role of phenylpyruvic acid in the pathophysiological mechanisms of phenylketonuria remains unknown.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalases protect cellular proteins from oxidative modification in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae cells had higher antioxidant enzyme activities under growth in ethanol than that in glucose as a carbon and energy source. The correlations between catalase activity and protein carbonyl level (r(2)=0.857), between catalase and glucose-6-phosphate dehydrogenase activities (r(2)=0.924) and between protein carbonyl levels and glucose-6-phosphate dehydrogenase activity (r(2)=0.988) under growth in ethanol were found. Growing in ethanol the strain deficient in cytosolic and peroxisomal catalases had 7.1-fold higher level of carbonyl proteins than that of wild-type strain. Our data suggest that in vivo catalases may protect glucose-6-phosphate dehydrogenase against oxidative inactivation.     

Relationship of the oxidative pentose shunt pathway to lipid synthesis in Drosophila melanogaster

The tissue activities of the oxidative pentose shunt enzymes, glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44), in the larvae of Drosophila melanogaster are not dependent on the amount of flux through the oxidative pentose shunt pathway. An oxidative pentose shunt deficiency effects about a 40% reduction in the NADPH concentration in early third instar larvae, resulting in a six-fold difference in the NADPH/NADP+ ratio between wild-type and pentose-shunt-deficient larvae. The capacity of pentose-shunt-deficient larvae to synthesize triglyceride in response to a high concentration of dietary sucrose is only 73% of the wild-type level. Environmental temperature influences on the fatty acid composition of larvae are not altered by an oxidative pentose shunt deficiency.     

Cooperativity between 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the lumen of the endoplasmic reticulum

The functional coupling of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase was investigated in rat liver microsomal vesicles. The activity of both enzymes was latent in intact vesicles, indicating the intraluminal localization of their active sites. Glucose-6-phosphate, a substrate for hexose-6-phosphate dehydrogenase, stimulated the cortisone reductase activity of 11beta-hydroxysteroid dehydrogenase type 1. Inhibition of glucose-6-phosphate uptake by S3483, a specific inhibitor of the microsomal glucose-6-phosphate transporter, decreased this effect. Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled glucose-6-phosphate, indicating the stimulation of hexose-6-phosphate dehydrogenase activity. A correlation was shown between glucose-6-phosphate-dependent cortisone reduction and cortisone-dependent glucose-6-phosphate oxidation. The results demonstrate a close cooperation of the enzymes based on co-localization and the mutual generation of cofactors for each other.     

Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer disease.

We analyzed glucose-6-phosphate dehydrogenase, the rate-controlling enzyme of the pentose phosphate pathway and free sulfhydryls, to study redox balance in Alzheimer disease. Glucose-6-phosphate dehydrogenase plays a pivotal role in homeostatic redox control by providing reducing equivalents to glutathione, the major nonenzymatic cellular antioxidant. There is a multitude of evidence that marks oxidative stress proximally in the natural history of Alzheimer disease. Consistent with a role for glutathione in defense against increased reactive oxygen, we found an upregulation of glucose-6-phosphate dehydrogenase together with increased sulfhydryls in Alzheimer disease. These data indicate that reductive compensation may play an important role in combating oxidative stress in Alzheimer disease.     

Glucose-6-phosphate dehydrogenase activity and NADPH/NADP+ ratio in liver and pancreas are dependent on the severity of hyperglycemia in rat

Hyperglycemia is associated with metabolic disturbances affecting cell redox potential, particularly the NADPH/NADP+ ratio and reduced glutathione levels. Under oxidative stress, the NADPH supply for reduced glutathione regeneration is dependent on glucose-6-phosphate dehydrogenase. We assessed the effect of different hyperglycemic conditions on enzymatic activities involved in glutathione regeneration (glucose-6-phosphate dehydrogenase and glutathione reductase), NADP(H) and reduced glutathione concentrations in order to analyze the relative role of these enzymes in the control of glutathione restoration. Male Sprague-Dawley rats with mild, moderate and severe hyperglycemia were obtained using different regimens of streptozotocin and nicotinamide. Fifteen days after treatment, rats were killed and enzymatic activities, NADP(H) and reduced glutathione were measured in liver and pancreas. Severe hyperglycemia was associated with decreased body weight, plasma insulin, glucose-6-phosphate dehydrogenase activity, NADPH/NADP+ ratio and glutathione levels in the liver and pancreas, and enhanced NADP+ and glutathione reductase activity in the liver. Moderate hyperglycemia caused similar changes, although body weight and liver NADP+ concentration were not affected and pancreatic glutathione reductase activity decreased. Mild hyperglycemia was associated with a reduction in pancreatic glucose-6-phosphate dehydrogenase activity. Glucose-6-phosphate dehydrogenase, NADPH/NADP+ ratio and glutathione level, vary inversely in relation to blood glucose concentrations, whereas liver glutathione reductase was enhanced during severe hyperglycemia. We conclude that glucose-6-phosphate dehydrogenase and NADPH/NADP+ were highly sensitive to low levels of hyperglycemia. NADPH/NADP+ is regulated by glucose-6-phosphate dehydrogenase in the liver and pancreas, whereas levels of reduced glutathione are mainly dependent on the NADPH supply.     

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Hemoglobin and red cell enzyme variation in some populations of the Republic of Vietnam with comments on the malria hypothesis

The blood of Vietnamese, Khmer, Cham, Rhade, Sedang and Stieng populations of the Republic of Vietnam was studied for hemoglobins, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase and adenylate kinase by starch gel electrophoresis. Plasmodium falciparum malaria parasite rates were obtained in all groups but the Stieng. The prevalence of glucose-6-phosphate dehydrogenase deficiency was lowest in the Sedang (0.004), the Vietnamese (0.014) and the Rhade (0.023). The highest prevalence of glucose-6-phosphate dehydrogenase deficiency was found in the Stieng (0.153). The lowest frequencies of hemoglobin^E were in the Vietnamese (0.025) and the Sedang (0.029). All other groups had high frequencies of hemoglobin^E, the highest being the Stieng (0.365). The prevalence of phosphogluconate dehydrogenase^B ranged from 0.000 in the Stieng to 0.054 in the Vietnamese. The Vietnamese were not differentiated from the Sedang at the glucose-6-phosphate dehydrogenase or the hemoglobin loci but were differentiated at the phosphogluconate dehydrogenase locus. Using the three markers most of the populations studied in South Vietnam were distinguishable one from the other. There was variable correlation between the frequency of hemoglobin^E and the prevalence of glucose-6-phosphate dehydrogenase deficiency. A correlation of the endemicity of falciparum malaria and the frequency of glucose-6-phosphate dehydrogenase deficiency and of hemoglobin^E was inconclusive. The frequency of the adenylate kinase² allele was low to absent. Adenylate kinase³ was found in the Khmer and in the Stieng.     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Correlation between the rate of protein and nucleic acid synthesis and the intensity of various glucose metabolic pathways in rat liver cells

A sharp and strong suppression of protein synthesis by cycloheximide in liver cells of starving rats is paralleled with activation of RNA synthesis and glucose-6-phosphate dehydrogenase production. Subsequent reconstitution and stimulation of protein synthesis (6-12 hrs after cycloheximide injection) result in activation of hexokinase. Upon stimulation of DNA synthesis (48-60 hrs after cycloheximide injection) the activity of both enzymes is very low. Since glucose-6-phosphate dehydrogenase appears to be the limiting step of glucose decay via the pentose phosphate pathway, and hexokinase is the limiting step of glycolysis, it was assumed that RNA synthesis predominantly occurs via the pentose phosphate pathway, while that of proteins via glycolysis.     

Radioimmunoassay and chemical properties of glucose-6-phosphate dehydrogenase and of a specific NADP(H)-binding protein (FX) from human erythrocytes

Two sensitive radioimmunoassays, based on a double-antibody technique, were developed which allow detection of nanogram amounts of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and of a so far unknown NADP(H)-binding protein present in human erythrocytes (designated FX).The two proteins isolated in homogeneous form from human erythrocytes were iodinated with ¹²⁵I by means of lactoperoxidase. Antisera to both purified proteins were raised in rabbits and sequentially adsorbed on human erythrocytes and on human serum before use. No cross-reaction between the two proteins was apparent.Hemolysates from normal as well as from glucose-6-phosphate dehydrogenase-deficient subjects were investigated for their content in both immunoreactive proteins using the two radioimmunoassay methods. This preliminary study showed significantly lowered levels of immunoreactive glucose-6-phosphate dehydrogenase in erythrocytes from subjects carrying the Mediterranean variant of this enzyme (characterized by severe deficiency of catalytic activity), compared with normal subjects. This figure was reversed as concerns the content of immunoreactive FX which was found to be twice as high in glucose-6-phosphate dehydrogenase Mediterranean erythrocytes as in normal ones.The two purified proteins were submitted to a comparative analysis of their chemical properties including NH₂-terminal residues, CNBr peptides and tryptic fingerprints. These studies revealed significant differences in the primary structures of the two proteins and therefore tend to exclude FX'x being a discrete product arising from degradation of native glucose-6-phosphate dehydrogenase. Moreover, amino axid analysis and tryptic fingerprints indicated that FX, as well as glucose-6-phosphatase dehydrogenase, is composed of very similar and possibly identical polypeptide chains.     

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Influence of very low doses of ionizing radiation on Synechococcus lividus metabolism during the initial growth phase

Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione     

Glycogen metabloism in the developing chick glycogen body: functional significance of the direct oxidative pathway.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase were quantitatively determined for the first time in glycogen body tissue from late embryonic and neonatal chicks. For comparative purposes, the activities of these enzymes were examined also in liver and skeletal muscle from pre- and post-hatched chicks. The present data show that both the embryonic and neonatal glycogen body lack glucose-6-phosphatase, but contain relatively high levels of glucose-6-phosphate dehydrogenase. The activity of each dehydrogenase in either embryonic or neonatal glycogen body tissue is two- to five-fold greater than that found in muscle or liver from pre- or post-hatched chicks. The relatively high activities observed for both dehydrogenases in the glycogen body, together with the absence of glucose-6-phosphatase activity in that tissue, suggest that the direct oxidative pathway (pentose phosphate cycle) of glucose metabolism is a functionally significant route for glycogen utilization in the glycogen body. It is hypothesized that the glycogen body is metabolically linked to lipid synthesis and myelin formation in the central nervous system of the avian embryo.     

The NAD+ precursors, nicotinic acid and nicotinamide upregulate glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase mRNA in Jurkat cells

To better understand the role of nicotinic acid and nicotinamide in the regulation of the oxidative stress response, we measured the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PD) mRNA in Jurkat cells treated with these NAD+ precursors. We used a modified nonradioactive Northern blot method and detected the mRNA using 18-mer digoxigenin (DIG)-labeled oligonucleotides as probes. We observed increased levels of the mRNAs for the two enzymes in treated cells. Our findings suggest that the NAD+ precursors may protect against oxidative stress and DNA damage by up-regulating the stress response genes GAPDH and G6PD.