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.     

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.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

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     

Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes

Historically, it has been theorized that the oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular gluthione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired to catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H₂O₂-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H₂O₂ challenge in the G6PD-deficient cell whiel only decrease was observed in normal cells. Supplmentation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P < 0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP⁺ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ration in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.     

Structure and function of glucose-6-phosphate dehydrogenase-deficient variants in Chinese population

A systematic study on the structure and function of Glucose-6-phosphate dehydrogenase (G6PD) variations was carried out in China. A total of 155,879 participants were screened for G6PD deficiency by the G6PD/6PGD ratio method and 6,683 cases have been found. The prevalence of G6PD deficiency ranged from 0 to 17.4%. With informed consent, 1,004 cases from 11 ethnic-based groups were subjected to molecular analysis. Our results showed the followings: (1) The G6PD variants are consistent across traditional ethnic boundaries, but vary in frequencies across ethnic-based groups in Chinese population, (2) The G6PD variants in Chinese population are different from those in African, European, and Indian populations, (3) A novel G6PD-deficiency mutation, 274C→T, has been found, and (4) Denaturing high performance liquid chromatography is of great advantage to detecting G6PD-deficient mutations for diagnosis and genetic counseling. Moreover, functional analysis of the human G6PD variants showed the following: (1) The charge property, polarity, pK-radical and side-chain radical of the substituting amino acid have an effect on G6PD activity, (2) The G6PDArg459 and Arg463 play important roles in anchoring NADP⁺ to the catalytic domain to maintain the enzymatic activity, and (3) The sequence from codon 459 to the carboxyl terminal is essential for the enzymatic function.     

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.     

Characterization of global metabolic responses of glucose-6-phosphate dehydrogenase-deficient hepatoma cells to diamide-induced oxidative stress

Glucose-6-phosphate dehydrogenase (G6PD) is crucial to NADPH generation and redox homeostasis. We have recently shown that G6PD deficiency predisposes cells to oxidant-induced cell death, and it is associated with the impairment of glutathione regeneration. It remains unclear what other metabolic pathways are affected by G6PD deficiency and whether the altered metabolism disturbs cellular redox homeostasis and underlies increased susceptibility to oxidants. In this study, we examined the effects of diamide on global metabolite profiles of SK-Hep1-derived SK-i-Gi and SK-i-Sc cells, which could inducibly express short hairpin RNA (shRNA) against G6PD (Gi) and control shRNA (Sc), respectively. There was no significant difference in their metabolite profiles under uninduced conditions. Doxycycline (Dox) addition resulted in over 70% decrease in G6PD activity in SK-i-Gi cells. This was accompanied by relatively minor changes in the metabolome of SK-i-Gi cells. Upon further diamide treatment, the metabolite profiles of both SK-i-Gi and SK-i-Sc cells changed in a time-dependent manner. A number of metabolic pathways, including those involved in energy metabolism and metabolism of amino acids and glutathione, were affected. However, the changes in the metabolite profile of Dox-treated SK-i-Gi cells were distinct from those of control cells (i.e., Dox-treated SK-i-Sc, SK-i-Gi, and SK-i-Sc cells). Cellular glutathione was depleted, whereas its disulfide form increased significantly in diamide, Dox-treated SK-i-Gi cells. Metabolites related to energy metabolism, such as AMP, ADP, and acetylcarnitine, increased to a greater extent in these cells than in diamide-treated control cells. In contrast, NAD and glutathione dropped to lower levels in SK-i-Gi cells than in control cells. The NAD⁺ depletion in SK-i-Gi cells was accompanied by a significant increase in NAD kinase activity. Targeted analyses revealed that NADP⁺ and NADPH increased significantly in diamide, Dox-treated SK-i-Gi cells compared with similarly treated control cells. Our results suggest that diamide induces oxidation and depletion of glutathione in SK-i-Gi cells under conditions of G6PD shRNA induction and subsequently induces conversion of NAD⁺ to NADP⁺ through enhanced NAD kinase activity. This may represent a compensatory mechanism to restore cellular NADPH reserve in G6PD-deficient cells. It is accompanied by alteration in pathways of cellular energy metabolism, such as glycolysis and β-oxidation.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.     

Catechin gallates are NADP+-competitive inhibitors of glucose-6-phosphate dehydrogenase and other enzymes that employ NADP+ as a coenzyme

Recent studies have shown that glucose-6-phosphate dehydrogenase (G6PD) is an effectual therapeutic target for metabolic disorders, including obesity and diabetes. In this study, we used in silico and conventional screening approaches to identify putative inhibitors of G6PD and found that gallated catechins (EGCG, GCG, ECG, CG), but not ungallated catechins (ECG, GC, EC, C), were NADP(+)-competitive inhibitors of G6PD and other enzymes that employ NADP(+) as a coenzyme, such as IDH and 6PGD.     

Artifacts imitating aging of glucose-6-phosphate dehydrogenase in human erythrocytes.

Data in the literature based on the technique of graded osomotic hemolysis have been re-evaluated. Differences were previously found in the glucose-6-phosphate dehydrogenase/hemoglobin ratio and in the heat stability of the enzyme in hemolysates of 'old' and 'young' cells. These differences were believed to be due to the aging of the enzyme. As the erythrocyte membrane acts as a molecular sieve under hypotonic conditions [cf. Cseke, E., Váradi, A., Szabolcsi, G., and Biszku, E. (1978) FEBS Lett. 96, 15--18], the hemolysate obtained when a fraction is lysed does not properly represent the content of the lysed cells. As hemoglobin is lost from cells which are not yet lysed, the enzyme/hemoglobin ratio is underestimated in 'old' cells and overestimated in 'young' cells. It is further shown that the observed differences in the heat stability of glucose-6-phosphate dehydrogenase in the fractions obtained by graded hemolysis are due to the presence of different concentrations of endogeneous NADP. Therefore the published data obtained by graded osmotic hemolysis do not prove the assumption that the enzyme is aging during the lifetime of the erythrocyte.     

Human erythrocyte glucose 6-phosphate dehydrogenase. Evidence for competitive binding of NADP and NADPH.

The tetrameric form of human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) was investigated in respect to interaction of coenzyme at its ron-structural sites. 1:N⁶-ethenoadenine dinucleotide phosphate (ε-NADP), although displaying a lower catalytic efficiency compared to NADP, showed identical binding patterns, i.e. four moles per tetramer with a K_diss of 1.0 μM. Furthermore, spectrofluorometric titrations with NADPH in the absence and in presence of varying NADP concentrations revealed a typically competitive mechanism of binding of NADP (four moles) and NADP (two moles) at the non-structural sites of the tetramer.