Further studies on sheep polymorphic erythrocyte diaphorase

Starch gel electrophoresis of sheep hemolysates revealed anodically faster, poly. morphic NADH/NADPH diaphorase (Dial) and slower NADH diaphorase (Dia2). Frequencies of alleles Dia1 F and Dia1 S for six sheep breeds in Czechoslovakia are given and efficacy for parentage control is discussed. A heterogeneity in Dia2 is caused by a prolonged storage of samples.     

Further data on the incidence and segregation of genetically determined electrophoretic variants of human red cell NADH diaphorase.

Human red cell NADH diaphorase isozyme patterns have been examined in 3,060 unrelated Australians of European origin, by starch gel electrophoresis. 26 people with variant isozyme patterns were encountered: 12 were phenotype Dia 2-1 and 13 were Dia 4-1. A new variant isozyme pattern (Dia 7-1) was identified. No variants were identified in 100 Melanesians and 70 Australian Aborigines.     

Red cell NADH diaphorase variants in Japanese

Human red cell NADH diaphorase isozyme patterns were examined in 5,046 healthy adult Japanese by starch gel electrophoresis. Twenty had Dia 2-1 and 3 had Dia 4-1 phenotypes. The incidence of Dia variants in patients with mental retardation, cerebral palsy, epilepsy and Down's syndrome was also examined and compared with that of healthy people. It was noticed that thin-layer isoelectric focusing on polyacrylamide gel was very useful for discriminating variant bands from 'aging bands'.     

Study of human red cell NADH diaphorase (Dia1) in the Italian population

A total of about 4,500 individuals from Northern, Central and Southern Italy have been analyzed for red cell NADH diaphorase. The results show that the Italians differ significantly (p less than 0.005) from the other examined populations of European origin by showing a higher frequency of the Dia2 allele (6.4%) and a lower frequency of other Dia variants (0.6%).     

Properties of glutamate dehydrogenase from developing maize endosperm

Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o₂) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca²⁺. Saturation curves for (NH₄)₂SO₄ and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca²⁺, but substrate inhibition occurred without Ca²⁺. The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca²⁺ and Mn²⁺, but not by Cu₂₊ and Mg²⁺.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

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.     

Inhibition of electron transport activities in mitochondria from avocado and pepper fruit by naturally occurring polyamines

The effects of the naturally occurring polyamines, spermine, putrescine, and spermidine were explored on mitochondrial state 3. state 4, and uncoupled respiration activities, ADP/O ratio, respiratory control ratio of pepper ( Capsicum annuum L. cv. Early Cal Wonder) and avocado ( Persea americana Mill. cv. Booth-8 or Simmonds) mitochondria oxidizing either succinate, external NADH, malate, α-ketoglutarate or tetramethyl- p -phenylenediamine. Abnormally high concentrations of spermine and spermidine such as might occur during chilling stress of these chilling-sensitive fruits were detrimental to several oxidase activities, especially to external NADH oxidase. State 3 respiration for NADH oxidase was inhibited more than 70% by 10 m M spermine. The spermine inhibition of uncoupled NADH oxidase was not reversed by the presence of divalent cations including Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ at concentrations up to 10 m M or by 100 m M KCl. The inhibition primarily affected the V_max. Other possible sites of polyamine interactions are discussed.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.     

Eigenschaften des Elektronentransportsystems von Mitochondrien des Protisten Acanthamoeba castellanii Neff. II. Reinigung und Eigenschaften der NADH-Akzeptor-Oxydoreduktase des Elektronentransportsystems

ZUSAMMENFASSUNG. Es wird die Reinigung einer mitochondrialen NADH-Akzeptor-Oxydoreduktase (EC. 1.6.99.3) über folgende Stufen beschrieben: fraktionierte Ammonsulfatfällung, Chromatographie an DEAE-Cellulose und Molekularsiebchromatographie an Sephadex G 100. Das gewonnene Enzympräparat erwies sich als elektrophoretisch rein. Die Michaeliskonstante für NADH als einziges Substrat liegt bei 6,9 × 10⁻⁵ M NADH. Ein ebenfalls isoliertes extramitochondriales Enzym underscheidet sich vom intramitochondrialen in drei voneinander unabhängigen Eigenschaften: Substratspezifität, elektrophoretiche Mobilität, Molekulargewicht. Das isolierte intramitochondriale Enzym verhält sich gegenüber Atmungsketteninhibitoren ähnlich wie isolierte Mitochondrien.
SYNOPSIS. Mitochondrial NADH reductase (EC. 1.6.99.3) was isolated from Acanthamoeba castellanii by the following steps: fractionation by (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, and chromatography on Sephadex G 100 columns. Purity of the enzyme preparation was demonstrated by electrophoresis. With NADH as the sole substrate, the K _m= 6.9 × 10⁻⁵ M NADH. Extramitochondrial enzyme, also isolated from the ameba, differed from the mitochondrial one in substrate specificity, electrophoretic mobility, and molecular weight. The mitochondrial NADH reductase and isolated mitochondria were affected in the same way by respiratory chain inhibtors.     

Properties of the glyceraldehyde-3-P dehydrogenase in heterocysts and vegetative cells of cyanobacteria

Abstract Glyceraldehyde-3-P dehydrogenase (GAPDH) in heterocysts and vegetative cells of 3 N₂-fixing cyanobacteria was found to utilize both NAD⁺ and NADP⁺. The enzyme activity was enhanced by thiols (glutathione, reduced lipoic acid and dithiothreitol). GAPDH of the 3 cyanobacterial species was not activated by thioredoxin. Heterocysts have now been shown to possess all the enzymes of glycolysis and the tricarboxylic acid cycle to convert glyceraldehyde-3-phosphate (GAP) to oxoglutarate and glutamate. The GAPDH reaction is a major source for the generation of NADH, which is oxidized by a thylakoid-bound NADH:plastoquinone oxidoreductase in heterocysts.     

Localization of alkaline phosphatase and NADH diaphorase in the principal cells of the guinea pig epididymis

The activities of alkaline phosphatase and reduced nicotinamide adenine dinucleotide (NADH) diaphorase in the principal cells of the guinea pig epididymis were studied histochemically. Alkaline phosphatase activity was absent from the principal cells but was present in the basement membrane of the epididymal epithelium. NADH diaphorase activity was distributed throughout the cytoplasm of the principal cells in each epididymal segment. There was a gradual increase in NADH diaphorase activity from segments 1 through 7. Possible functions of alkaline phosphatase and NADH diaphorase in the epididymis are discussed.     

1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l⁻¹ at D = 0·15 h⁻¹), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD ⁺ and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD ⁺ content in the mutants as well as in the wild type.     

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.     

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.     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)⁻¹min⁻¹] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca²⁺, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent K_m (SHAM) of ca 40 μ M (with NADH). The dependence of O₂ consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H₂O₂ was produced for every 1 mol O₂ consumed. Endogenous catalase converted this H₂O₂ to O₂ and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O₃. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed.     

Demonstration of HOQNO and antimycin A sensitive coupling of NADH oxidation and APS and sulfite reduction in a marine Desulfovibrio strain

Abstract In cell suspensions of the marine sulfate-reducing bacterium Desulfovibrio 20020 (DSM 3099) permeabilized with formaldehyde or Triton X-100, sulfite-dependent NADH oxidation activities of 0.05 μmol · min⁻¹· mg⁻¹ protein were detected. NADH oxidation coupled to APS, thiosulfate and fumarate reduction was also demonstrated. All the activities were subject to inhibition by HOQNO and antimycin A. The rate of NADH oxidation coupled to the reduction of sulfite was extremely low in cell-free extracts. The physiological function and possible mechanism of the NADH oxidation coupled to the reduction of various electron acceptors are discussed.     

THE EFFECT OF MODERATE AND MARKED HYPERCAPNIA UPON THE ENERGY STATE AND UPON THE CYTOPLASMIC NADH/NAD+ RATIO OF THE RAT BRAIN

Abstract— The energy state of brain tissue was evaluated from the tissue concentrations of ATP, ADP and AMP and the cytoplasmic NADH/NAD⁺ ratio from the tissue, CSF and blood concentrations of lactate and pyruvate, and from the intracellular pH', in rats exposed to carbon dioxide concentrations of 640 per cent. The hypercapnia had no significant effect on the energy state of the tissue. Hypercapnia of increasing severity gave rise to a progressive decrease in the pyruvate concentration; the lactate concentration fell at low CO₂ concentrations, but no further decrease was observed at CO₂ concentrations greater than 20 per cent. There was a progressive rise in the intracellular lactate/pyruvate ratio at increasing CO₂ concentrations, corresponding to the fall in intracellular pH, i.e. the calculated NADH/NAD⁺ ratios remained normal. It is therefore concluded that hypercapnia does not affect the cytoplasmic redox state.     

Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.