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.     

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.     

Erythrocyte glucose-6-phosphate dehydrogenase activity in Brazilian monkeys

Activities of glucose-6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase as well electrophoretic mobility of glucose-6-phosphate dehydrogenase from erythrocytes of Brazilian monkeys were investigated. Glucose-6-phosphate dehydrogenase activity of simian was 4 times higher than the human values. Regarding electrophoretic studies, the results, did not reveal any intraspecific polymorphism. A comparison of erythrocyte glucose-6-phosphate dehydrogenases among primates is also presented.     

Amino acid sequence of the carboxy-terminal end of human erythrocyte glucose-6-phosphate dehydrogenase

Human erythrocyte glucose-6-phosphate dehydrogenase was purified to homogeneity by a simplified procedure, consisting of 2',5'-ADP-Sepharose affinity chromatography, followed by Sephadex G-100 gel filtration. The carboxy-terminal region of the protein was identified by carboxypeptidase digestion: the sequence -Lys-Leu-COOH was found instead of the reported -Gly-COOH, thus showing identity with the carboxy-terminal sequence of glucose-6-phosphate dehydrogenase from human leukocytes and platelets. In addition, the carboxyl-terminal peptide was isolated from a tryptic digest of the protein and sequenced. The sequence is: Trp-Val-Asp-Pro-His-Lys-Leu.     

Human erythrocyte glucose-6-phosphate dehydrogenase. Identification of a reactive lysyl residue labelled with pyridoxal 5'-phosphate

Human erythrocyte glucose-6-phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5'-phosphate. The binding of one mole of pyridoxal 5'-phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose-6-phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate-binding site. A tryptic peptide containing the pyridoxal-5'-phosphate-binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose-6-phosphate dehydrogenase amino acid sequence. Comparison with glucose-6-phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme. The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose-6-phosphate dehydrogenase.     

Oxidative inactivation of the calcium-stimulated neutral proteinase from human red blood cells by divicine and intracellular protection by reduced glutathione

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.     

NADP-binding proteins causing reduced availability and sigmoid release of NADP+ in human erythrocytes

Glucose-6-phosphate dehydrogenase catalyzes the initial and rate-limiting step of the pathway that is the principal source of NADPH in many cells. Earlier studies of cells from several species indicated that the intracellular enzyme is under severe and unexplained restraint or inhibition. Moreover, the intracellular enzyme of human erythrocytes exhibits sigmoid kinetics, whereas the purified enzyme exhibits only classical kinetics. We here report that most of the NADP in the human erythrocyte is bound by soluble proteins. In addition, the fraction of unbound NADP that is in the oxidized form, [NADP+]/[NADP], varies in a sigmoid manner relative to the fraction of bound NADP that is in the oxidized form. These features of intracellular binding of NADP: 1) account for the previously unexplained inhibition and sigmoid kinetics of glucose-6-phosphate dehydrogenase within human erythrocytes and 2) represent a system in which activity of a rate-limiting enzyme is largely determined by the binding and release of substrate and product by intracellular proteins other than the enzyme itself.     

Plasmodium falciparum: thiol status and growth in normal and glucose-6-phosphate dehydrogenase deficient human erythrocytes

Thiol status and growth in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Experimental Parasitology 57, 239-247. The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamide-treated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria.     

Regulation of glucose-6-phosphate dehydrogenase activity during caloric restriction in human adipose tissue

Glucose-6-phosphate dehydrogenase activity was significantly lower in adipose tissue of human subjects after 7 days of severe caloric restriction on low-carbohydrate diets and had returned to normal values 4 days after the subjects resumed normal diets. Three other enzyme activities (malate-NADP dehydrogenase, oxaloacetate decarboxylating; ATP-citrate lyase and 6-phosphofructokinase) were not significantly affected by these dietary changes. These results are consistent with separate control of glucose-6-phosphate dehydrogenase activity versus other 'lipogenic' enzyme activities in human adipose tissue.     

Heat-labile enzymes in circulating erythrocytes of a progeria family.

Cultured skin fibroblasts from subjects with progeria contain an increased fraction of heat-labile enzymes and other altered proteins. To determine whether freshly obtained cells are similarly affected, erythrocytes from a progeric female and her clinically normal parents were analyzed for heat-lability of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Hemolysates of the child's whole erythrocyte populations and young erythrocytes isolated by equilibrium density centrifugation contained significantly higher heat-labile fractions of both enzymes compared to control hemolysates. Values in both parents were intermediate to those of their daughter and controls, consistent with autosomal recessive inheritance in this family. The primary source of these multiple protein defects is unknown but may reside in a mutant gene producing abnormal protein turnover or defective DNA repair. An increased fraction of thermolabile enzymes in circulating erythrocytes should be useful in identifying persons at risk for progeria and other disorders of premature aging.     

Variations of Some Enzyne and Coenzyme Concentrations in Nornal Human Erythrocytes Fractionated by Velocity Sedimentation

Abstract

The concentrations of glucose-6-phosphate dehydrogenase, adenosine triphosphate and 2,3-diphosphoglycerate were measured in various fractions of normal human erythrocytes separated by velocity sedimentation in isotonic saline and sucrose buffer based on their tendency to aggregate. The levels of 2,3-diphosphoglycerate varied most drastically in fresh erythrocytes, being lowest in fast sedimenting cells and highest in slowly sedimenting cells. This result implies that the 2,3-diphosphoglycerate concentrations is not constant in all normal human erythrocytes and that 2,3-diphosphoglycerate may be involved in the mechanism of red cell clumping in vitro.     

Glucose 6-phosphate dehydrogenase activity in membranes of erythrocytes from normal individuals and subjects with Mediterranean G6PD deficiency

Unsealed, hemoglobin-free erythrocyte ghosts contain low yet significant levels of Glucose 6-phosphate dehydrogenase (G6PD) activity. This activity is comparable in erythrocyte ghosts obtained from normal individuals and from G6PD-deficient subjects (of Mediterranean type), in spite of the marked differences found in the corresponding cytosolic compartments. The membrane preparations can bind purified human G6PD (type B) to their cytoplasmic surface according to patterns of positive cooperativity. 2.4 × 10⁴ and 1.6 × 10⁴ G6PD-binding sites are present on the inner surface of each ghost obtained from normal and from G6PD-deficient erythrocytes, respectively, the relevant association constants being 2.8 × 10⁶ M^?1 and 0.82 × 10⁶ M^?1. The interaction of G6PD with the ghosts is unaffected by different ionic strengths or by metabolites such as glucose 6-phosphate, NADP and NADPH.     

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

Evaluation of effect of some corticosteroids on glucose-6-phosphate dehydrogenase and comparative study of antioxidant enzyme activities

Corticosteroids are anti-inflammatory drugs that are similar to the natural corticosteroid hormones produced by the cortex of the adrenal glands. The objective of this study was to scrutinize effects of some corticosteroids on glucose-6-phosphate dehydrogenase (G6PD) and some antioxidant enzymes. Initially, G6PD was purified from human erythrocytes by using ammonium sulphate precipitation and affinity chromatography. The two drugs, dexamethasone phosphate and prednisolone, investigated on the purified enzyme inhibited the enzyme activity. Comparative in vivo studies were performed to determine the effects of dexamethasone phosphate on the antioxidant enzyme activities using Spraque-Dawley rats. G6PD and catalase (CAT) activities were found significantly lower than in the control, whereas glutathione peroxidase (GP) activity was significantly increased in the erythrocytes of rats the receiving drug; glutathione reductase (GR) activity was unaffected. The results imply that dexamethasone phosphate may affect oxidative stress by changing antioxidant enzyme activities.     

Sigmoid kinetics of human erythrocyte glucose-6-phosphate dehydrogenase

Several disagreements and inconsistencies have appeared regarding whether human erythrocyte glucose-6-phosphate dehydrogenase exhibits sigmoid or classical kinetics with respect to NADP+ binding. The latest report is that the purified enzyme exhibits classical kinetics while the intracellular enzyme exhibits sigmoid kinetics (H. N. Kirkman, and G. F. Gaetani (1986) J. Biol. Chem. 261, 4033-4038). The various investigations were carried out at fixed pH, ionic strength, and temperature. The steady-state kinetics of crude and purified erythrocyte glucose-6-phosphate dehydrogenase are reported here at various temperatures, ionic strengths, and pH values and as a function of glucose 6-phosphate concentration. Sigmoid kinetics were observed for both purified and crude enzyme samples at high pH, temperature, ionic strength, and concentration of glucose 6-phosphate with Hill coefficients varying between 1.40 and 1.90. In contrast, at low pH, temperature, and ionic strength, the crude enzyme samples exhibit sigmoid kinetics while the purified samples exhibit classical kinetics despite the high concentration of glucose 6-phosphate. High concentrations of glucose 6-phosphate and factors favoring the enzyme in the dimeric form are necessary conditions for the observation of sigmoid kinetics in human erythrocyte glucose-6-phosphate dehydrogenase. These factors are high pH, ionic strength, and temperature. The observed sigmoid kinetics in this enzyme is explained as arising from tetramer-dimer transitions.     

Purification of glucose 6-phosphate dehydrogenase from Buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo (Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2('),5(')-ADP-Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K(M) and V(max) values for NADP(+) and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K(i) values and the type of inhibition were determined by means of Lineweaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH.     

Glucose-6-phosphate dehydrogenase (G6PD) Iserlohn and G6PD Regensburg: two new severe enzyme defects in German families

Two new inheritable variants of glucose-6-phosphate dehydrogenase have been found in two unrelated German families. Patients with one variant (G6PD Iserlohn, also referred to as G6PD I) suffered from intermittent hemolytic crises caused by fava beans; patients with the other variant (G6PD Regensburg, G6PD II) disclosed chronic nonspherocytic hemolytic anemia aggravated by drug treatment. Due to their unusual biochemical characteristics, the new variants were designated G6PD Iserlohn and G6PD Regensburg. Both variants showed a reduction of enzyme activity to about 6% of the normal in erythrocytes, normal electrophoretic mobility, increased affinity for glucose-6-phosphate, a reduced affinity for NADP and a pH optimum in the neutral region (7.0 and 7.5). G6PD Iserlohn had a decreased affinity for the inhibitor NADPH; G6PD Regensburg had a normal inhibitor constant. Deamino NADP was utilized at an increased rate by G6PD Regensburg. G6PD Iserlohn was thermostable, G6PD Regensburg mildly instable. G6PD activity in leukocytes was normal in G6PD Iserlohn and reduced to the same degree as in erythrocytets in G6PD Regensburg. The cause of the decreased activity of G6PD Iserlohn appears to be in vivo instability; in G6PD Regensburg further mechanisms might include reduced specific activity or reduced synthesis of the variant enzyme.     

Protein methylation as a marker of aspartate damage in glucose-6-phosphate dehydrogenase-deficient erythrocytes: role of oxidative stress.

The 'Mediterranean' variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme L-isoaspartate (d-aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl-labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification. We present here evidence that, in G6PD-deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t-butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid-reactive substances. L-Isoaspartate residues increased dramatically in G6PD-deficient erythrocytes in response to such treatment, compared with baseline conditions. The increased susceptibility of G6PD-deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.     

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.