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.     

Malarial parasite hexokinase and hexokinase-dependent glutathione reduction in the Plasmodium falciparum-infected human erythrocyte

The metabolism of glucose in Plasmodium falciparum-infected human erythrocytes is increased 50- to 100-fold. This is accomplished in part by parasite-directed synthesis of a protozoan hexokinase with unique kinetic, electrophoretic, and heat stability properties. The total hexokinase activity is increased approximately 25-fold over that of control uninfected erythrocytes of the same age from the same donor. The parasite hexokinase has a lower affinity for glucose than the mammalian enzyme (Km = 431 microM +/- 21 S.D. for the parasite enzyme versus 98 microM +/- 10 for the erythrocyte enzyme), but the Km for ATP and the Vmax for both glucose and ATP are similar. The NADPH-dependent reduction of oxidized glutathione (GSSG) requires the formation of glucose 6-phosphate which in turn is metabolized by the pentose shunt pathway in which NADPH is generated. Using glucose as the substrate, lysates of P. falciparum-infected normal erythrocytes demonstrated enhanced ability to reduce GSSG. The rate of GSSG reduction was proportional both to the parasitemia and the hexokinase activity of the lysates. However, infected glucose-6-phosphate dehydrogenase-deficient red cell lysates displayed a severely restricted ability to reduce GSSG under the same conditions. In conclusion, P. falciparum-infected red cells contain a parasite-encoded hexokinase with unique properties which initiates the large increase in glucose consumption. In normal infected red cells, reduction of GSSG is also dependent upon hexokinase activity, but in infected glucose-6-phosphate dehydrogenase-deficient red cells, the absence of this pentose shunt enzyme remains the rate-limiting step in GSSG reduction.     

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes.

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

Impairment of the calcium pump of human erythrocytes by divicine

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocytes membranes at 50-100 microM concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.     

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65–70% of the rate with NADH. Added cytochrome b₅ stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b₅ and cytochrome b₅ reductase determine the rate of methemoglobin reduction in hemolysates.     

Author index

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65–70% of the rate with NADH. Added cytochrome b₅ stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b₅ and cytochrome b₅ reductase determine the rate of methemoglobin reduction in hemolysates.     

The transport of oxidized glutathione from the erythrocytes of various species in the prescence of chromate

1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were not inhibited. 4. The GSSG formed was transported from the erythrocytes to the medium. 5. The transport rate of GSSG from glucose 6-phosphate dehydrogenase-deficient erythrocytes subjected to hydrogen peroxide diffusion in the presence of chromate was comparable with that from normal and glucose 6-phosphate dehydrogenase-deficient erythrocytes. 6. The rate of transport of GSSG from erythrocytes of various species studied could be ranked: pigeon>rabbit>rat>donkey>man>dog>horse>sheep>chicken>fish.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37 degrees C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H2O2 and HbO2, may be related to the pathogenesis of hemolysis in favism.     

Regulation of the human-erythrocyte hexose-monophosphate shunt under conditions of oxidative stress. A study using NMR spectroscopy, a kinetic isotope effect, a reconstituted system and computer simulation

The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 1H NMR and a biochemical assay were used to measure the changes. A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose-6-phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate-limiting step of oxidatively stimulated flux through the hexose monophosphate shunt. The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [1-2H]glucose replaced glucose as the source of reducing equivalents, showed that glucose-6-phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6-bisphosphate and glycerate 2,3-bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6-bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed. A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37°C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H₂O₂ and HbO₂, may be related to the pathogenesis of hemolysis in favism.     

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.     

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.     

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.     

The role of glutathione reductase in the interplay between oxidative stress response and turnover of cytosolic NADPH in Kluyveromyces lactis

The phosphoglucose isomerase mutant of the respiratory yeast Kluyveromyces lactis (rag2) is forced to metabolize glucose through the oxidative pentose phosphate pathway and shows an increased respiratory chain activity and reactive oxygen species production. We have proved that the K. lactis rag2 mutant is more resistant to oxidative stress (OS) than the wild type, and higher activities of glutathione reductase (GLR) and catalase contribute to this phenotype. Resistance to OS of the rag2 mutant is reduced when the gene encoding GLR is deleted. The reduction is higher when, in addition, catalase activity is inhibited. In K. lactis, catalase activity is induced by peroxide-mediated OS but GLR is not. We have found that the increase of GLR activity is correlated with that of glucose-6-phosphate dehydrogenase (G6PDH) activity that produces NADPH. G6PDH is positively regulated by an active respiratory chain and GLR plays a role in the reoxidation of the NADPH from the pentose phosphate pathway in these conditions. Cytosolic NADPH is also used by mitochondrial external alternative dehydrogenases. Neither GLR overexpression nor induction of the OS response restores growth on glucose of the rag2 mutant when the mitochondrial reoxidation of cytosolic NADPH is blocked.     

Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.

The effect of oxidative stress on human red blood cell AMP-deaminase activity was studied by incubating either fresh erythrocytes or hemolysates with H(2)O(2) (0.5, 1, 2, 4, 6, 8, and 10 mm) or NaNO(2) (1, 5, 10, 20, and 50 mm), for 15 min at 37 degrees C. AMP-deaminase tremendously increased by increasing H(2)O(2) or NaNO(2) at up to 4 and 20 mm, respectively (maximal effect for both oxidants was 9.5 and 6.5 times higher enzymatic activity than control erythrocytes or hemolysates, respectively). The incubation of hemolysates with iodoacetate (5-100 mm), N-ethylmaleimide (0.1-10 mm), or p-hydroxymercuribenzoate (0.1-5 mm) mimicked the effect of oxidative stress on AMP-deaminase, indicating that sulfhydryl group modification is involved in the enzyme activation. In comparison with control hemolysates, changes of the kinetic properties of AMP-deaminase (decrease of AMP concentration necessary for half-maximal activation, increase of V(max), modification of the curve shape of V(o) versus [S], Hill plots, and coefficients) were recorded with 4 mm H(2)O(2)- and 1 mm N-ethylmaleimide-treated hemolysates. Data obtained using 90% purified enzyme, incubated with Fenton reagents (Fe(2+) + H(2)O(2)) or -SH-modifying compounds, demonstrated that (i) reactive oxygen species are directly responsible for AMP-deaminase activation; (ii) this phenomenon occurs through sulfhydryl group modification; and (iii) the activation does not involve the loss of the tetrameric protein structure. Results of experiments conducted with glucose-6-phosphate dehydrogenase-deficient erythrocytes, challenged with increasing doses of the anti-malarial drug quinine hydrochloride and showing dramatic AMP-deaminase activation, suggest relevant physiopathological implications of this enzymatic activation in conditions of increased oxidative stress. To the best of our knowledge, this is the first example of an enzyme, fundamental for the maintenance of the correct red blood cell energy metabolism, that is activated (rather than inhibited) by the interaction with reactive oxygen species.     

Glucose-dependent Protection by Dietary Selenium against Haemolysis of Rat Erythrocytes <Emphasis Type="Italic">in vitro</Emphasis>

THE occurrence in man of drug-induced haemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes¹ suggested the possibility of an analogy to the haemolysis which occurs in vitamin E deficient red blood cells. Cohen and Hochstein² have shown that haemolysis in G6PD deficient cells is associated with the inability of the cell to generate adequate reduced glutathione (GSH) through GSSG reductase because of the impaired generation of NADPH. Moreover, there is evidence that glucose protects red blood cells from haemolysis by its ability to provide NADPH through G6PD which subsequently generates GSH³. The G6PD deficient cell, however, cannot maintain an adequate concentration of GSH in the cell, even in the presence of glucose⁴, whereas the normal cell can maintain a normal concentration of GSH in the presence of glucose, preserving the integrity of the red blood cell. Vitamin E protects red blood cells from haemolysis whether supplied in vivo or in vitro and its effect has usually been demonstrated without glucose in the incubation medium. Although selenium prevents many of the same deficiency symptoms as vitamin E, it has not been uniformly effective in preventing the in vitro haemolysis of red blood cells. If a protective action of selenium against haemolysis were dependent on the presence of GSH, or if selenium were involved in the generation of GSH, selenium would not be expected to prevent haemolysis unless glucose was present in the incubation medium to provide a constant source of NADPH for the generation of GSH from GSSG through GSSG reductase (Fig. 1).     

Transient inhibition of glucose utilization by erythrocytes during the acute stage of typhoid fever

The exact reason for hemolysis of glucose-6-phosphate dehydrogenase-deficient (G6PD) erythrocytes in patients with typhoid fever is unknown. Therefore, glucose utilization by normal and G6PD-deficient erythrocytes was measured during incubation with plasma of healthy controls as well as from patients in acute or recovery stages of typhoid fever. Glucose utilization in normal and G6PD-deficient erythrocytes significantly decreased compared to the controls when incubated with plasma of patients with acute typhoid fever, which normalized to the baseline after recovery from typhoid fever, suggesting an acquired alteration in G6PD enzyme properties by Salmonella typhi or its endotoxins.     

The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.