NAD(P)H Dehydrogenase-Dependent, Antimycin A-Sensitive Electron Donation to Plastoquinone in Tobacco Chloroplasts

Cyclic electron transport around PSI through the NAD(P)H dehydrogenasecomplex (NDH) in tobacco leaf disks, measured as an increasein the dark level of Chl fluorescence after the onset of darkness,was inhibited by antimycin A, an inhibitor of ferredoxin quinonereductase (FQR), suggesting that antimycin A inhibits not onlythe FQR-mediated cyclic flow but also the NDH-dependent flow.This electron flow was inhibited also by amytal, an inhibitorof mitochondrial NDH and by nigericin. The reduction of plastoquinonewas detected when NADPH and ferredoxin were added to the suspensionof the osmotically ruptured chloroplasts of the wild type andNDH-defective mutant. Because the addition of NADPH alone didnot induce the reduction, membrane-bound ferredoxin NADP⁺reductase(FNR) was supposed to reduce ferredoxin, which may be a moredirect electron donor for the plastoquinone reduction. The presenceof two types of reducing enzymes was suggested from the bi-phasicinhibition of plastoquinone reduction by antimycin A in thewild type. It is proposed that the reducing activity inhibitedby antimycin A at a low concentration is attributed to FQR andthe less sensitive activity to NDH. (Received June 29, 1998; Accepted September 7, 1998)     

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts

Intact chloroplasts from spinach showed a transient increasein Chl fluorescence after saturating illumination with actiniclight and its yield depended on the duration of illuminationand the intensity of the actinic light (AL). The increase waspartially suppressed when antimycin A was added immediatelyafter termination of the AL. The inhibited fluorescence increase,therefore, reflected the electron flow from the reductant(s)that had accumulated during the actinic illumination to theplastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitiveCyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743].Addition of dihydroxyacetone phosphate (DHAP) to chloroplastscaused the enhancement of the increase in fluorescence afterAL, which was inhibited by antimycin A. Decay of the transientlyraised fluorescence was retarded by 2-heptyl-4-hydroxyquinolineN-oxide and stigmatellin, suggesting that re-oxidation of thereduced PQ pool is coupled with the operation of Q-cycle. Althoughthe activity of the stromal enzyme system that supplies NADPHon addition of DHAP was constant irrespective of light or darkness,the capacity of the intact chloroplasts to show a DHAP-dependentfluorescence increase had a limited lifetime after AL was turnedoff. This result suggests that the antimycin A-sensitive Cytb-559 or ferredoxin-NADP reductase is activated by light anddeactivated in the dark. In ruptured chloroplasts, the additionof NADPH increased the dark fluorescence yield only in the presenceof Fd, which also was inhibited by antimycin A. Thus the photoregulatorymechanism of Cyt b-559 (Fd) in intact chloroplasts appearedto be lost when chloroplasts were ruptured. (Received June 21, 1995; Accepted September 25, 1995)     

Kinetics of NADPH-Induced Non-Photochemical Reduction of the Plastoquinone Pool in Spinach Chloroplasts

Kinetics of non-photochemical reduction of the photosynthetic intersystem electron transport chain by exogenous NADPH was examined in osmotically lysed spinach chloroplasts by chlorophyll (Chl) fluorescence measurements under anaerobic condition. Upon the addition of NADPH, the apparent F₀ increased sigmoidally, and the value of the maximal slope was calculated to give the reduction rate of plastoquinone (PQ) pool. Application of 5 µM antimycin A lowered significantly both the ceiling and the rate of the NADPH-induced Chl fluorescence increase, while the suppressive effect of 10 µM rotenone was slighter. This indicated that dark reduction of the PQ pool by NADPH in spinach chloroplasts under O₂-limitation condition could be attributed mainly to the pathway catalysed sequentially by ferredoxin-NADP⁺ oxidoreductase (FNR) and ferredoxin-plastoquinone reductase (FQR), rather than that mediated by NAD(P)H dehydro- genase (NDH).     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Enhanced NAD(P)H:Quinone Reductase Activity Prevents Glutamate Toxicity Produced by Oxidative Stress

Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.     

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H₂O₂ could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H₂O₂-generating enzyme. This increase in PGA formation due to H₂O₂ was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O₂ and by the NAD(P)H-oxidizing system.     

集胞蓝藻PCC6803含疏水亚基的NAD（P）H脱氢酶亚复合体的分离

利用离子交换与凝胶过滤层析 ,从n dodecylβ D maltoside(DM)处理的集胞蓝藻SynechocystisPCC6 80 3细胞粗提液中 ,首次分离到两个包含NDH疏水亚基NdhA的亚复合体。酶活性分析表明 ,分离到的NDH亚复合体具有NADPH 氮蓝四唑 (NBT)氧化还原酶活性 ,以NADPH为电子供体可以还原铁氰化钾、二溴百里香醌 (DBMIB)、二氯酚靛酚 (DCPIP)、duroquinone以及UQ 0等质醌类电子受体。     

Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane     

Nonenzymatic reduction of nitrosobenzene to phenylhydroxylamine by NAD(P)H

The nonenzymatic reduction of nitrosobenzene by NADPH and NADH in aqueous buffer solution at 25°C is described. Both reactants quantitatively convert nitrosobenzene to phenylhydroxylamine. Rate constants for reduction (k_r) were determined spectrophotometrically and found to be identical at pH 5.7 and 7.4 and independent of buffer concentration. The values of k_NADH (124–149 M^?1 sec^?1) and k_NADPH (131–170 M^?1 sec^?1) are essentially identical. The reaction is not subject to general catalysis or specific salt effects. The oxidation of phenylhydroxylamine by NAD(P) to nitrosobenzene is only stimulated by a factor of 1.2 over oxidation in its absence (when the ratio of NADP: phenylhydroxylamine was 8:1).     

Purification,Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Regulation of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

Activation of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3-phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3-phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time-course of activation appears to depend upon the actual concentration of 1,3-bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The K_a values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the K_a value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP-GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered K_avalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP-GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.     

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.     

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O₂ evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.