Mechanisms of ascorbic acid recycling in human erythrocytes.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.     

Adenine and pyridine nucleotides in the erythrocyte of different mammalian species

Adenine (ATP, ADP, AMP) and pyridine nucleotides (NADP+, NADPH, NAD+, NADH) concentrations have been determined by HPLC in the erythrocytes from five different mammalian species (pig, rat, mouse, rabbit and cow) and compared to those in human red blood cells. Two different extraction procedures have been used and the results obtained are compared and discussed. A good correlation between the different abilities of the erythrocytes of the six species to utilize glucose and the NAD+/NADH ratio was found, with high NAD+/NADH ratio in the red blood cell of the species with high glucose utilization rates. The levels of all the glycolytic enzymes and some of the pentose phosphate shunt enzymes were also determined.     

Rat alveolar macrophages require NADPH for superoxide production in the respiratory burst. Effect of NADPH depletion by paraquat

Alveolar macrophages can be stimulated by concanavalin A to produce extracellular superoxide. Conflicting opinions exist, however, concerning the relative importance of the oxidation of either NADPH or NADH in the generation of (Formula: see text) by surface membrane-stimulated phagocytic cells. Alveolar macrophages were obtained from adult male rats by lavage with phosphate-buffered saline. Cells (approximately 10(6)/ml) were incubated in Krebs-Ringer phosphate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and ferricytochrome c for 15 min at 37 degrees C before addition of concanavalin A. Release of (Formula: see text) was detected as the difference in cytochrome c reduction, followed at 550 nm, in the absence and presence of superoxide dismutase. Superoxide production by concanavalin A-stimulated alveolar macrophages was markedly increased in the presence of glucose but fructose, lactate, and pyruvate were without effect. Paraquat (methylviologen), an oxidation-reduction dye, significantly reduced concanavalin A-stimulated (Formula: see text) production when incubated at 1 mM with alveolar macrophages in the absence of glucose. The effect of paraquat was reversed by glucose, but fructose, lactate, and pyruvate could not reverse paraquat inhibition. Paraquat enhanced oxidation of NADPH (but not NADH) by cell supernatant and increased pentose phosphate shunt activity in resting macrophages, but did not affect mitochondrial respiration or ATP content of alveolar macrophages. These results suggest that paraquat is able to specifically deplete NADPH in alveolar macrophages while not affecting NADH or ATP. Our conclusion is that NADPH is essential for the production of (Formula: see text) by concanavalin A-stimulated alveolar macrophages.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

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.     

Kernel abortion in maize : I. Carbohydrate concentration patterns and Acid invertase activity of maize kernels induced to abort in vitro

The distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C₃-plants, pea and leek, and two C₄-plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue.     

Evidence for a role of taurine in the in vitro oxidative toxicity of neutrophils toward erythrocytes

Production of hydrogen peroxide and secretion of myeloperoxidase by stimulated neutrophils resulted in myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl), the reaction of HOCl with taurine to yield taurine monochloramine (TauNHCl), and accumulation of TauNHCl in the extracellular medium. When erythrocytes were present, the yield of TauNHCl was lower as the result of uptake of TauNHCl into erythrocytes. The zwitterion taurine was not taken up, but the anion TauNHCl and other anionic oxidants including taurine dichloramine (TauNCl2) and L-alanine chloramines were transported into erythrocytes by the anion-transport system. Oxidation of intracellular components such as glutathione (GSH) by taurine chloramines resulted in reduction of the chloramines and trapping of taurine within erythrocytes. At high oxidant:erythrocyte ratios, TauNHCl also oxidized hemoglobin (Hb) and depleted ATP, but caused little lysis. TauNCl2 was much more effective as a lytic agent. At low oxidant:erythrocyte ratios, the chloramines caused net loss of GSH when no glucose was provided, but Hb was not oxidized and GSH content returned to normal when glucose was added. Therefore, anionic chloramines may mediate oxidative toxicity when the neutrophil:erythrocyte ratio is high. Under more physiologic conditions, chlorination of taurine by neutrophils and the uptake and reduction of TauNHCl by erythrocytes prevents accumulation of oxidants and may protect blood cells, plasma components, and tissues against oxidative toxicity.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Divergent effects of different oxidants on glutathione homeostasis and protein damage in erythrocytes from diabetic patients: effects of high glucose.

Long-term complications of diabetes mellitus have been ascribed to both the effects of prolonged hyperglycemia and to increased oxidative stress. In an attempt to identify the mechanisms underlying the acute effects of hyperglycemia on oxidative stress, we investigated the hypothesis that high glucose might lead to an insufficiency in reducing equivalents (such as NADPH) and thus to a disruption in the glutathione-dependent antioxidant defences and to an incapacity to deal with oxidant attack. For this purpose, erythrocytes from diabetic patients were incubated for 0-90 min in 5.55 or 33.3 mM D-glucose containing tertbutyl hydroperoxide 0.5 and 1 mM, Menadione 100 microM, or glucose oxidase. The time course of the changes in non-protein bound glutathione (reduced and oxidised), lactate and pyruvate, alanine and fluorescent products of oxidative proteolysis, hemolysis and methemoglobin was monitored. The results show that although glucose utilisation was unaffected, all oxidants caused a persistent decrease in total non-protein-bound glutathione suggesting binding to proteins. However, changes in glutathione and redox status differed between the various oxidants and were not directly related to the extent of oxidative cellular damage. In these experimental conditions, with short incubations and using the erythrocyte as the simplest cellular model of glucose metabolism, neither high glucose nor the diabetic condition worsened the susceptibility of erythrocytes to acute in vitro oxidative damage.     

Transient Changes in the Energy State of Adenylates and the Redox States of Pyridine Nucleotides in Chlorella Cells Induced by Environmental Changes

The unicellular green algae Chlorella ellipsoidea was used tostudy transient changes in the energy state of adenylates andthe redox states of pyridine nucleotides induced by environmentalchanges. The transition from anaerobic to aerobic conditionsin the dark induced a sharp rise in the ATP ratio [ATP/(ATP+ADP+AMP)],a sudden decrease in the NADH ratio [NADH/(NAD⁺+NADPH)] anda transient drop in the NADPH ratio [NADPH/(NADP⁺+NADPH)]. Illuminationafter a dark period under anaerobic, CO₂-free conditions inducedsharp increases in the ATP and NADPH ratios and a slower decreasein the NADH ratio. Illumination under aerobic conditions, ineither the presence or absence of CO₂, caused a sharp increasein the NADPH ratio, a small increase in the ATP ratio and aslower increase in the NADH ratio. In the presence of CO₂, asubsequent large drop in the NADPH ratio occurred. Darkeningunder anaerobic, CO₂-free conditions induced a sudden decreasein the ATP ratio, a temporary fall in the NADPH ratio and aslow increase in the NADH ratio. Darkening under aerobic conditionsinduced transient drops in the ATP and NADPH ratios and a suddendrop in the NADH ratio. The addition of CO₂ to the atmospherewith illumination produced a decrease in all three parameters. These results are discussed in relation to current theoriesof the interaction between photosynthesis and respiration. Ourobservations indicate that the energy and reducing potentialsgenerated by photochemical processes are used for and controlother processes besides CO₂ fixation in photosynthetic cells. (Received December 3, 1981; Accepted May 4, 1982)     

Regulation of pathways of glucose metabolism in kidney: Specific linking of pentose phosphate pathway activity with kidney growth in experimental diabetes and unilateral nephrectomy

The pentose phosphate pathway operates at an elevated level in rat kidney following induction of diabetes and in the compensatory hypertrophy following unilateral nephrectomy in control and alloxan-diabetic rats, as shown by the yields of ¹⁴Co₂ from [1-¹⁴C]glucose, [6-¹⁴C]glucose and ³H₂O yields from [2-³H]glucose. The elevated flux through the pentose phosphate pathway is correlated with the increased RNA content and weight of the kidney. The direct utilization of NADPH for reductive synthetic reactions and the potential for indirect utilization via the sorbitol route and the linked transhydrogenase reactions of the glucuronate-xylulose pathway, for NADH and ATP generation, are also discussed.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells

This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.     

Distinct effects on the secretion of MTRAP and AMA1 in Plasmodium yoelii following deletion of acylated pleckstrin homology domain-containing protein

Plasmodium, the causative agents of malaria, are obligate intracellular organisms. In humans, pathogenesis is caused by the blood stage parasite, which multiplies within erythrocytes, thus erythrocyte invasion is an essential developmental step. Merozoite form parasites released into the blood stream coordinately secrets a panel of proteins from the microneme secretory organelles for gliding motility, establishment of a tight junction with a target naive erythrocyte, and subsequent internalization. A protein identified in Toxoplasma gondii facilitates microneme fusion with the plasma membrane for exocytosis; namely, acylated pleckstrin homology domain-containing protein (APH). To obtain insight into the differential microneme discharge by malaria parasites, in this study we analyzed the consequences of APH deletion in the rodent malaria model, Plasmodium yoelii, using a DiCre-based inducible knockout method. We found that APH deletion resulted in a reduction in parasite asexual growth and erythrocyte invasion, with some parasites retaining the ability to invade and grow without APH. APH deletion impaired the secretion of microneme proteins, MTRAP and AMA1, and upon contact with erythrocytes the secretion of MTRAP, but not AMA1, was observed. APH-deleted merozoites were able to attach to and deform erythrocytes, consistent with the observed MTRAP secretion. Tight junctions were formed, but echinocytosis after merozoite internalization into erythrocytes was significantly reduced, consistent with the observed absence of AMA1 secretion. Together with our observation that APH largely colocalized with MTRAP, but less with AMA1, we propose that APH is directly involved in MTRAP secretion; whereas any role of APH in AMA1 secretion is indirect in Plasmodium.     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

The pentose phosphate pathway in relation to fat synthesis in the developing castor oil seed

Slices of 25- to 28-day-old developing castor bean endosperm were incubated with various ¹⁴C- and ³H-labeled substrates to determine the amount of glucose dissimilated in the pentose phosphate pathway and to determine the use of the reduced nucleotides so produced in fatty acid synthesis. Ten to 12% of the metabolized glucose traversed the pentose phosphate pathway, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) production would be sufficient to supply 51 to 68% of the reducing equivalents required for fat synthesis. However, using ³H-NADPH produced from 3-³H-glucose as a tracer, it was found that only 40% of the NADPH produced in the pentose phosphate pathway was used in fat synthesis. Thus the actual contribution of the reducing equivalents generated from the pentose phosphate pathway to fat synthesis was 20 to 27% of that required. Because of the methods and assumptions, this value represents a minimal estimate of NADPH used in fat synthesis, and the actual contribution may be somewhat higher. However, tritium from ³H-NADH generated from 1-³H-ethanol was incorporated into fatty acids, and it is contended that NADH may supply a large proportion of the reducing equivalents necessary for fat synthesis in this tissue.     

Isolation and some characteristics of glutathione reductase from rabbit erythrocytes (38548).

Glutathione reductase from rabbit erythrocytes was pruified to homogeneity and found to be a monomer with a mol wt of 60,000. Both NADPH and HADH were capable of acting as cofactors for the reduction of GSSG and the following kinetic values were obtained: Km, GSSG = 120 muM; Km, NADPH = 37 muM; Vmax = 23 mumoles NADPH/min/mg protein, Km, NADH = 420 muM; Vmax = 3 mumoles NADH/min/mg protein. Rabbit erythrocyte GR exhibited substrate inhibition, and was susceptible to inhibition by p-hydroxymercuribenzoate under certain conditions.