Interrelationship between aminopyrine oxidation and gluconeogenesis in hepatocytes prepared from fructose-pretreated mice

Aminopyrine oxidation was studied in isolated hepatocytes prepared from 24-h-starved mice (i) after induction of the NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase, but not the mixed function oxygenases by fructose, (ii) after induction of both mixed function oxygenases and NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase by phenobarbital and (iii) without any pretreatment. Phenobarbital pretreatment, as expected, increased the rate of aminopyrine oxidation of isolated hepatocytes. However, fructose pretreatment also enhanced the rate of N-demethylation of aminopyrine by more than 100% supporting the view that the availability of NADPH is rate limiting in drug oxidation under certain conditions. The role of malic enzyme and glucose-6-phosphate dehydrogenase in the NADPH supply for aminopyrine oxidation was investigated by the addition of two groups of gluconeogenic precursors: lactate or alanine and glycerol or fructose with the simultaneous measurement of glucose synthesis and aminopyrine N-demethylation. There was a clear correlation between the increased rate of aminopyrine oxidation and the decreases of glucose production caused by aminopyrine. Gluconeogenesis in the presence of 1 mM aminopyrine was decreased by 70-80% when alanine or lactate were used as precursors, it was decreased by only 35-40% when glucose production was started from glycerol or fructose; in an accordance with the facts that NADPH generation and gluconeogenesis starting from alanine or lactate share two common intermediates--malate and glucose-6 phosphate--, while there is only one common intermediate--glucose-6 phosphate--if fructose or glycerol are used. Similar results were obtained with the addition of the structurally dissimilar hexobarbital. It is concluded that besides malic enzyme, glucose-6-phosphate dehydrogenase also takes part in NADPH supply for drug oxidation in glycogen-depleted hepatocytes.     

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.     

Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

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.     

Comparison of ratios of covalent binding to total metabolism of the pulmonary toxin, 4-ipomeanol, In vitro in pulmonary and hepatic microsomes,and the effects of pretreatments with phenobarbital or 3-methylcholanthrene

Studies of the ratios of the amounts of 4-ipomeanol covalently bound to the total amounts metabolized support the view that the high rates of $\text{in}\text{vitro}$ pulmonary microsomal alkylation by 4-ipomeanol reflect high rates of NADPH-mediated metabolic activation of the compound rather than a relative deficiency of a microsomal detoxication pathway. Moreover, the ability of 3-methylcholanthene pretreatment, but not phenobarbital pretreatment, to shift the $\text{in}\text{vivo}$ target organ alkylation and toxicity of 4-ipomeanol from the lung to the liver in rats could not be explained by a major alteration in the balances between microsomal toxication and detoxication pathways measurable in the $\text{in}\text{vitro}$ systems examined, nor upon a major change in the nature of the reactive 4-ipomeanol metabolites produced in the lungs or livers of the pretreated animals.     

Quantitative requirements for NADPH in the support of aromatization by human placental microsomes

Suitable incubation conditions were developed for reduced pyridine nucleotide protection and regeneration to permit quantitative assessment of the NADPH requirement for steroid aromatization by human placental microsomes. 10 mM dithiothreitol was found to protect NADP(H) from microsomal nucleotide pyrophosphatase and 2 mM nicotinamide mononucleotide was utilized to control nucleotide glycohydrolase activity. Under these assay conditions, the initial rates of aromatization obtained with restricted NADPH levels were critically dependent upon both the amount and the source of exogenous NADPH-regenerating dehydrogenase system. With excess $\text{Leuconostoc mesenteroides}$ glucose-6-phosphate dehydrogenase, an apparent Km for NADPH of 0.20 μM was observed for aromatization which is significently below all previous estimates of the NADPH requirement and which is at greatest only one-tenth the Km value for NADPH utilization by NADPH-cytochrome $\text{c}$ reductase. These findings suggest a potential regulatory role for both NADPH-generating and NADPH-accepting enzymes in the support of estrogen biosynthesis.     

Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean?

NADPH cytochrome c (cyt c) reductase and glucose-6-phosphatase, two enzymes thought to be restricted to the endoplasmic reticulum (ER) and widely used as ER markers, are present in isolated Golgi fractions assayed immediately after their isolation. Both enzymes are rapidly inactivated in fractions stored at 0 degrees C in 0.25 M sucrose, conditions which do not affect the activity of other enzymes in the same preparation. The inactivation process was shown to be dependent on time and protein concentration and could be prevented by EDTA and catalase. Morphological evidence shows that extensive membrane damage occurs parallel with the inactivation. Taken together with the immunological data in the companion paper, the findings indicate that the enzymes NADPH cyt c reductase and probably glucose-6-phosphate are indigenous components of Golgi membranes.     

The effects of carbon tetrachloride on rat liver microsomes during the first hour of poisoning in vivo, and the modifying actions of promethazine

The effects of an oral administration of carbon tetrachloride on various liver microsomal and supernatant components were studied 1hr. and 2hr. after dosing. The modifications of such early changes resulting from a concomitant administration of promethazine together with the carbon tetrachloride were also investigated. The microsomal components studied were: cytochromes P-450 and b(5); inorganic pyrophosphatase; NADH- and NADPH-cytochrome c reductases; NADH- and NADPH-neotetrazolium reductases; a lipid-peroxidation system associated with the oxidation of NADPH and stimulated by ADP and Fe(2+). NAD- and NADP- DT-diaphorases were measured in the supernatant solution remaining after isolation of liver microsomes, and the distribution of RNA phosphorus between the microsomes and supernatant solution was also determined. Carbon tetrachloride produced a rapid fall in inorganic pyrophosphatase activity, a rather slower decrease in cytochrome P-450 content of the microsomes and small increases in the activities of NADH-cytochrome c reductase and neotetrazolium reductases. The activities of NADPH-cytochrome c reductase, the NADPH-ADP/Fe(2+)-linked lipid-peroxidation system, DT-diaphorases and the content of cytochrome b(5) in the microsomes were unchanged. There was also a loss of RNA phosphorus from the microsomes into the supernatant solution. The RNA phosphorus redistribution, the decrease in inorganic pyrophosphatase and the increases in neotetrazolium reductase activities were at least partially prevented by a concomitant dosing with promethazine. However, the decrease in cytochrome P-450 was not affected by promethazine treatment. These early changes are discussed in terms of the liver necrosis produced by carbon tetrachloride and which is greatly retarded in its onset by the administration of promethazine.     

Glucose-6-phosphate oxidation pathway in rat-liver microsomal vesicles. Stimulation under oxidative stress

The glucose-6-phosphate oxidation pathway present in microsomes was studied using intact microsomal membranes. The oxidation activity, which was measured by monitoring the formation of 14CO2 from [1-14C]glucose 6-phosphate, was greatly stimulated when azodicarboxylic acid bis(dimethylamide), methylene blue or cumene hydroperoxide was added to the assay mixture. Glutathione peroxidase and glutathione reductase are suggested to be involved in the oxidation reaction induced by these oxidizing reagents. We detected a significant activity of the glutathione reductase inherent to microsomes. The microsomal glutathione reductase is latent and requires detergent to reveal its activity. 4,4'-Diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) inhibited the 14CO2 formation, but the inhibition was released by the addition of a detergent. Moreover, the inhibitory effect of DIDS was reversed by glucose 6-phosphate but not by mannose 6-phosphate. We conclude that the glucose-6-phosphate oxidation pathway in intact microsomes starts working under oxidative stress and that a transporter specific for glucose 6-phosphate is involved in the reaction.     

Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

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.     

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.     

Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship.

The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.     

Maintenance of luminal NADPH in the endoplasmic reticulum promotes the survival of human neutrophil granulocytes

The present study demonstrates the expression of hexose-6-phosphate dehydrogenase and 11 beta-hydroxysteroid dehydrogenase type 1 in human neutrophils, and the presence and activity of these enzymes in the microsomal fraction of the cells. Their concerted action together with the previously described glucose-6-phosphate transporter is responsible for cortisone-cortisol interconversion detected in human neutrophils. Furthermore, the results suggest that luminal NADPH generation by the cortisol dehydrogenase activity of 11 beta-hydroxysteroid dehydrogenase type 1 prevents neutrophil apoptosis provoked by the inhibition of the glucose-6-phosphate transporter. In conclusion, the maintenance of the luminal NADPH pool is an important antiapoptotic factor in neutrophil granulocytes.     

Enzyme activities of the primary and secondary metabolism of simultaneously permeabilized and immobilized plant cells

After various permeabilization procedures, plant cells obtained from suspension cultures of Catharanthus roseus are permeable to enzyme substrates which cannot enter the intact cell. Five enzymes of the primary metabolism, hexokinase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, malic enzyme, and citrate dehydrogenase, are studied with special emphasis on the two-enzyme system hexokinase/glucose-6-phosphate dehydrogenase. It is found that permeabilized cells immobilized in agarose retain their enzymatic activities far longer on storage than nonimmobilized cells. Whereas cells permeabilized by various methods show different initial enzymatic activity, the subsequent decrease of activity with time is at the same relative rate. Optimal initial activity is found with dimethyl sulfoxide-treated plant cells. As an enzyme of the secondary metabolism, we choose cathenamice reductase forming ajmalicine alkaloids from cathenamine. It is found that in dimethyl sulfoxide-treated cells the enzyme activity remains intact and that the addition of the coenzyme required in this step, NADPH, considerably increases the yield of product formed. Also, excretion into the medium is enhanced in both these immobilized and permeabilized systems.     

Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue

The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.     

Successive purification of several enzymes having affinities for phosphoric groups of substrates by affinity chromatography on P-cellulose.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyruvate kinase of Candida utilis and baker's yeast, when in anionic form, were adsorbed on a cation exchanger, P-cellulose, due to affinities similar to those for the phosphoric groups of their respective substrates; thus, glucose-6-phosphate dehydrogenase was readily eluted by either NADP+ or NADPH, glutathione reductase by NADPH, 6-phosphogluconate dehydrogenase by 6-phosphogluconate, and pyruvate kinase by either ATP or ADP. This type of chromatography may be called "affinity-adsorption-elution chromatography"; the main principle is different from that of so-called affinity-elution chromatography. Based on these findings, a large-scale procedure suitable for successive purification of several enzymes having affinities for the phosphoric groups of their substrates was devised. As an example, glucose-6-phosphate dehydrogenase was highly purified from baker's yeast and crystallized.     

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.