The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation

The redox level and compartmentation of pyridine nucleotides was studied under photorespiratory and non-photorespiratory conditions using rapid fractionation of barley ( Hordeum vulgare L. cv. Gunilla, Svalöv) leaf protoplasts. From comparative measurements of the NADPH/NADP⁺ ratio and the ATP/ADP ratio one acidic and one alkaline extraction medium was chosen which quenched the metabolism very efficiently. The mitochondrial NADH/NAD⁺ was higher under photorespiratory conditions than under non-photorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, lowered the mitochondrial NADH/NAD⁺ ratio. This supports the hypothesis that glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol. The chloroplastic NADPH/NADP⁺ as well as the NADH/NAD⁺ ratios were quite stable in saturating and limiting CO₂ as well as in the presence of aminoacetonitrile, although the triosephosphate/phosphoglycerate ratios changed. Thus, the redox level in the stroma seems to be tightly regulated.     

Changes in the level of oxidized and reduced pyridine nucleotides during cold acclimation of winter rape plants

Prolonged cold (2°C) treatment of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górczański) markedly modified the pattern of leaf growth and brought about changes in the level of pyridine nucleotides already during the first few days of treatment. The NAD⁺, NADP⁺ and NADPH levels markedly increased but there was practically no effect on the NADH level. Changes in the respective nucleotide levels were reflected by changes in anabolic and catabolic reduction charges. The former increased by 70%, whereas the latter decreased by 44%. Alterations in pyridine nucleotide levels and the reduction charges are discussed in terms of possible mechanisms involved, as well as in terms of their role in plant adaptation to cold.     

Pyridine Nucleotide Changes In Hepatocytes Exposed To Quinones

Quinones may be toxic by a number of mechanisms. including arylation and oxidative stress caused by redox cycling. Using isolated hepatocytes, we have studied the cytotoxicity of four quinones. with differing abilities to arylate cellular nucleophiles and redox cycle. in relation to their effects on cellular pyridine nucleotides. High concentrations of menadione (redox cycles and arylates). 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) 2.3-dimethoxy-1.4-naphthoquinone (a pure redox cycler) and p-benzoquinone (a pure arylator) caused an initial decrease in NAD⁺ and loss of viability, which was not prevented by 3-aminobenzamide. an inhibitor of poly(ADP-ribose)polymerase. In contrast. 3-aminobenzamide inhibited the loss of NAD' and viability caused by dimethyl sulphate so implicating poly(ADP-ribose)polymerase in its toxicity but not that of the quinones. Non-toxic concentrations of menadione. 2.3-dimethoxy-1.4-naphthoquinone and 2-hydroxy-1.4-naphthoquinone all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD⁺ was accompanied by a small. transient increase in NADP⁺ and followed by a larger. prolonged increase in NADPH and total NADP⁺ + NADPH. Nucleotide changes were not observed with non-toxic concentrations of p-benzoquinone. Our findings suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides. in an attempt to combat the effects of oxidative stress     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775

Abstract Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h⁻¹ to 0.5 h⁻¹, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.     

Menadione- (2-methyl-1,4-naphthoquinone-) dependent enzymatic redox cycling and calcium release by mitochondria

The results presented in this paper reveal the existence of three distinct menadione (2-methyl-1,4-naphthoquinone) reductases in mitochondria: NAD(P)H:(quinone-acceptor) oxidoreductase (D,T-diaphorase), NADPH:(quinone-acceptor) oxidoreductase, and NADH:(quinone-acceptor) oxidoreductase. All three enzymes reduce menadione in a two-electron step directly to the hydroquinone form. NADH-ubiquinone oxidoreductase (NADH dehydrogenase) and NAD(P)H azoreductase do not participate significantly in menadione reduction. In mitochondrial extracts, the menadione-induced NAD(P)H oxidation occurs beyond stoichiometric reduction of the quinone and is accompanied by O2 consumption. Benzoquinone is reduced more rapidly than menadione but does not undergo redox cycling. In intact mitochondria, menadione triggers oxidation of intramitochondrial pyridine nucleotides, cyanide-insensitive O2 consumption, and a transient decrease of delta psi. In the presence of intramitochondrial Ca2+, the menadione-induced oxidation of pyridine nucleotides is accompanied by their hydrolysis, and Ca2+ is released from mitochondria. The menadione-induced Ca2+ release leaves mitochondria intact, provided excessive Ca2+ cycling is prevented. In both selenium-deficient and selenium-adequate mitochondria, menadione is equally effective in inducing oxidation of pyridine nucleotides and Ca2+ release. Thus, menadione-induced Ca2+ release is mediated predominantly by enzymatic two-electron reduction of menadione, and not by H2O2 generated by menadione-dependent redox cycling. Our findings argue against D,T-diaphorase being a control device that prevents quinone-dependent oxygen toxicity in mitochondria.     

Decrease of NADH in HeLa cells in the presence of transferrin or ferricyanide

The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.     

A Hydrazine Coupled Cycling Assay Validates the Decrease in Redox Ratio under Starvation in Drosophila

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD⁺/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP⁺ and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD⁺ and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.     

Iron stress-induced redox reactions in bean roots

Iron stress-induced and constitutive redox activity of bean ( Phaseolus vulgaris L. cv. Delinel) roots was measured on intact plants using FeEDTA and ferricyanide as electron acceptors. The presence of the translation inhibitor cycloheximide caused a decrease in the reduction of both oxidants. However, a differential decline in the reduction rates of FeEDTA and ferricyanide was observed, suggesting enzyme heterogeneity. In the presence of the H⁺ -ATPase inhibitor vanadate, the reduction of FeEDTA was nearly completely suppressed in both Fe-deficient (–Fe) and Fe-sufficient (+Fe) plants, providing evidence for an involvement of plasma membrane-bound ATPase activity in the regulation of the reduction process. The inhibition of the ferricyanide reduction by vanadate was restricted to –Fe plants.
The data are interpreted in terms of simultaneous operation of distinct redox systems in roots of iron-deficient bean plants. The role of proton extrusion in iron stress-induced electron transfer is discussed.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH, NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8-9 U mg^-1 protein (µmoles mg^-1 min^-1) for NADPH and 3-4 U mg^-1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^-1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^-1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Thansplasmalemma redox reactions and ion transport in photosynthetic and heterotrophic plant cells

Extracellular ferricyanide reduction, NADH and ferrocyanide oxidation were investigated by spectrophotometrical method on photosynthetic freshwater plants ( Elodea canadensis Rich., Vallisneria spiralis L., Nitella flexilis L.) and heterotrophic tissues (roots of Triticum vulgare L., Hordeum vulgare L., Zea mays L., Pisum sativum L., Avena sativa L., Allium sativa L., Allium cepa L.). All species had ferricyanide reductase activity. The roots of land plants also carried out extracellular oxidation of NADH and ferrocyanide in contrast to leaves of the freshwater plants. External NADH stimulated ferricyanide reductase activity, but only with those objects that had external NADH oxidase activity. In all species ferricyanide decreased the membrane potential (MP), decreased the membrane resistance measured at a fixed current and inhibited K⁺ influx measured by flame photometry. The factors affecting ferricyanide reductase activity also influenced the inhibitory effect of ferricyanide on the MP and K⁺ transport. These results demonstrate a connection between transport, electrogenic and redox functions of the plasmalemma.     

Vacuolating cytotoxic activity of Pasteurella multocida causing haemorrhagic septicaemia in buffalo and cattle

Abstract Sequence data had indicated that cyanobacteria might possess a bidirectional hydrogenase with properties similar to the soluble enzymes from Alcaligenes eutrophus, Nocardia opaca and Desulfovibrio fructosovorans . The present study shows that extracts from the cyanobacterium Anacystis nidulans catalyse NAD(P)H-dependent H₂ evolution with low but significant activity and uptake of the gas with NAD(P)⁺ as the electron acceptor. NAD⁺ is the preferred electron acceptor and NADH the preferred donor compared to NADP⁺ and NADPH, respectively. Activity levels of this NAD(P)⁺dependent, bidirectional hydrogenase are too low to support chemoautotrophic growth in A. nidulans .     

Regulation of nicotinamide adenine dinucleotide phosphate levels in yeast

The steady-state levels and redox states of pyridine nucleotide pools have been studied in yeast as a function of external growth conditions. Yeast grown aerobically on 0.8% glucose show two distinct phases of logarithmic growth, a first phase utilizing glucose with ethanol accumulation, and a second phase utilizing ethanol. During growth on glucose, the size of the NADP pool (NADP⁺ + NADPH) is maintained at approximately 12% the size of the NAD pool (NAD⁺ + NADH). Upon exhaustion of glucose, the mechanism(s) that maintain the levels of NADP relative to NAD are altered, resulting in a rapid 2- to 2.5-fold decrease in the size of the NADP pool relative to the size of the NAD pool. The lower levels of NADP are maintained during growth on ethanol. The NAD pool is approximately 50% NADH during both the glucose and ethanol phases of growth, while the NADP pool is approximately 67 and 48% NADPH during the glucose and ethanol phases of growth, respectively. Rapid media transfer experiments show that the decrease in NADP is reversible, that it does not require the net synthesis of pyridine nucleotide or protein, and that changes in the size of the NADP pool relative to the total pyridine nucleotide pool are correlated with changes in the redox state of the NADP pool.     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.     

NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane studied using inside-out submitochondrial particles

Submitochondrial particles (SMP) were isolated from potato ( Solanum tuberosum L. cv. Bintje) tubers. The SMP were 91% inside-out and they were able to form a membrane potential, as monitored by oxonol VI, with succinate, NADH and NADPH. The pH dependence and kinetics of NADH and NADPH oxidation by these SMP was studied using three different electron acceptors – O₂, duroquinone and ferricyanide. In addition, the SMP were solubilized, fractionated by non-denaturing polyacrylamide gel electrophoresis, and the gels were stained for NAD(P)H dehydrogenase activity and specificity at different pH using Nitro Blue Tetrazolium. From the results we conclude that there are at least two distinct NAD(P)H dehydrogenases on the inner surface of the inner membrane: (1) Complex 1 which oxidizes NADH and deamino-NADH in a rotenone-sensitive manner, (O₂ as acceptor) with optimum activity at pH 8 and a very low K_m(NADH) of 3 μ M . It also oxidizes NADPH and deamino-NADPH in a rotenone-sensitive manner, but with a pH optimum at pH 5.8 and a very high K_m(NADPH) of more than 1 m M . This complex is found as a broad, diffuse band at the top of the gels. (2) A second dehydrogenase which oxidizes NADH in a rotenone-insensitive manner with optimum activity at pH 6.2 and a higher K_m(NADH) of 14 μ M . It also oxidizes NADPH in a rotenone-insensitive manner with an activity optimum at pH 6.8 and low K_m(NADPH) of 25 μ M . This dehydrogenase does not oxidize deamino-NAD(P)H. One of the sharp bands around the middle of the native gels may be caused by this dehydrogenase indicating that it has a relatively low molecular mass compared to Complex I. Several other NAD(P)H dehydrogenase bands were observed on the gels which we cannot yet assign.     

THE ENERGETICS OF EXTRACELLULAR FE(III) REDUCTION BY IRON-LIMITED CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Fe-limited cells of the green alga Chlamydomonas reinhardtii (Fe-limited growth rate = 0.3 d⁻¹) reduced extracellular Fe(III) to Fe(II) when Fe(III) was supplied as ferricyanide or Fe(III)-EDTA; Fe(III) reduction was stimulated by light. In both darkness and during photosynthesis, ferricyanide reduction was accompanied by a decrease in cellular NADPH levels, with a concomitant increase in NADP⁺. NADH and NAD⁺ levels were not measurably altered during ferricyanide reduction. Furthermore, cellular hexose monophosphate levels declined and 6-phosphogluconate levels increased during ferricyanide reduction. Levels of most glycolytic and tricarboxylic acid cycle intermediates were mostly unaltered. Ferricyanide reduction was also associated with a decrease in cellular ATP levels, a concomitant increase in ADP and AMP, and increased extracellular acidification. The acidification was sensitive to inhibition by the H⁺-ATPase inhibitor N,N' -dicyclohexylcarbodiimide (DCCD). We conclude that the oxidative pentose phosphate pathway provides reducing equivalents for Fe(III) reduction in darkness and also contributes reducing equivalents to Fe(III) reduction during photosynthesis. The decline in ATP was likely due to activation of the plasma membrane H⁺-ATPase during ferricyanide reduction and was not directly associated with provision of reducing equivalents.     

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.