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.     

Modelling NADH turnover in plant mitochondria

NADH is central to the functioning of mitochondrial respiration. It is produced by enzymes in, or associated with, the tricarboxylic acid cycle in the matrix, and it is oxidized by two respiratory chain enzymes in the inner membrane, the rotenone-sensitive complex I and the rotenone-insensitive internal NADH dehydrogenase (ND_in). A simplified kinetic model for NADH turnover in the matrix of plant mitochondria is presented. Only the two main NADH-producing enzymes, NAD-malate dehydrogenase [EC 1.1.1.37] (MDH) and NAD-malic enzyme [EC 1.1.1.39] (ME), are considered. This model reproduces the complex behaviour of malate oxidation by isolated mitochondria in response to additions of ADP (state 3/state 4), NAD⁺ and/or rotenone, as well as to changes in pH. It is found that MDH always operates at or close to equilibrium. Changes in the activity of complex I, ND_in, or ME are predicted to cause clear changes in the pattern of malate oxidation. In general, the model predicts high sensitivity to changes in the ME activity. In contrast, MDH activity can be reduced 100-fold without detectable changes in malate oxidation. It is demonstrated that it is not the high activity, but the equilibrium properties of MDH that are important for the redox-buffering function of MDH in the mitochondrial matrix. Binding of NAD⁺ and NADH in the matrix reduces the concentrations of free NAD⁺ and NADH, depending on the concentration of binding sites and the binding strength. On the basis of the modelling results it is estimated that a significant proportion of the mitochondrial NAD is bound.     

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.     

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Mitochondria isolated from cotyledons of dark-grown cucumber ( Cucumber sativus L., cv. Shimotsuki-Aonaga) seedlings after illumination with continuous far-red light showed an increased capacity for oxidation of malate or α-ketoglutarate, as compared with those from cotyledons of non-illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD⁺ had no effect on the rate of the oxidation of α-ketoglutarate or malate by mitochondria isolated from far-red light-treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light-treated ones. The NAD (NAD⁺+ NADH) content was higher in mitochondria isolated from continuously far-red light-treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far-red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.     

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.     

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.     

Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure

The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles - RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)^-1min^-1 for mitochondria, CO_2; fixation 46.8 μmol (mg Chi)^-1 h^-1 for chloroplasts.
Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD⁺, EC 1.2.4.1), pyruvate formate-lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH- and NADPH-dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction.     

Effects of cold on NAD+ kinase activity in winter rape plants

Low temperature (2°C) caused an increase in the activity of NAD⁺ kinase (EC 2.7.1.23) in leaves of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górezański). The enzyme activity markedly increased between day 4 and 11 of plant exposure to cold, then tended to decrease. Changes in activity of NAD⁺ kinase coincided with the previously observed changes in the levels of pyridine nucleotides, NADP(H) (U. Maciejewska and A. Kacperska, Physiol. Plant. 69: 687–691, 1987). As a result of cold treatment, Ca²⁺–calmodulin–dependent and Ca²⁺–calmodulin–independent NAD⁺ kinase activities increased to almost the same extent. It seems therefore, that the cold–induced activation of NAD⁺ kinase does not depend on the Ca²⁺–calmodulin complex.     

Continuous measurement of oxaloacetate in purified mitochondria from the leaves of Kalanchoë blossfeldiana

A new method for the continuous assay of oxaloacetate released or taken up by plant mitochondria during malate oxidation is described. It is based on the continuous spectrophotometric recording of the reduction level of externally added NAD⁺ (0.4 m M ) to a mitochondrial preparation. In the presence of 20 m M malate and of externally added malate dehydrogenase (EC 1.1.1.37), an equilibrium is reached instantaneously, bringing about a partial reduction of NAD⁺ and the production of a proportional amount of oxaloacetate (OAA). Owing to the presence of a very active OAA carrier on the inner mitochondrial membrane, the concentration at the equilibrium position of the reactants of the external MDH is permanently displaced by the OAA released or taken up by the mitochondria. Therefore, changes in OAA concentration can be followed from the measurement of the reduction level of the external NAD⁺. This method appears as sensitive as the classical enzymatic method, but is much more rapid and requires much less mitochondrial protein. The proposed method was applied to Percoll-purified mitochondria from the leaves of a CAM plant, Kalanchoë blossfeldiana Poelln. cv. Tom Thumb. The simultaneous recording of the change in OAA concentration and of oxygen uptake during malate oxidation emphasizes the major control exerted by OAA on the rate of malate oxidation.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2, confers enhanced tolerance to oxidative stress in Arabidopsis plants

Mutant pqr-216 from an Arabidopsis activation-tagged line showed a phenotype of increased tolerance to oxidative stress after treatment with 3 μ m paraquat (PQ). Based on the phenotype of transgenic plants overexpressing the genes flanking the T-DNA insert, it was clear that enhanced expression of a Nudix (nucleoside diphosphates linked to some moiety X) hydrolase gene, AtNUDX2 (At5g47650), was responsible for the tolerance. It has been reported that the AtNUDX2 protein has pyrophosphatase activities towards both ADP-ribose and NADH ( Ogawa et al ., 2005 ). Interestingly, the pyrophosphatase activity toward ADP-ribose, but not NADH, was increased in pqr-216 and Pro _35S :AtNUDX2 plants compared with control plants. The amount of free ADP-ribose was lower in the Pro _35S :AtNUDX2 plants, while the level of NADH was similar to those in control plants under both normal conditions and oxidative stress. Depletion of NAD⁺ and ATP resulting from activation of poly(ADP-ribosyl)ation under oxidative stress was observed in the control Arabidopsis plants. Such alterations in the levels of these molecules were significantly suppressed in the Pro _35S :AtNUDX2 plants. The results indicate that overexpression of AtNUDX2 , encoding ADP-ribose pyrophosphatase, confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from maintenance of NAD⁺ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions.     

ELECTROCONVULSIVE SEIZURE: AN INVESTIGATION INTO THE VALIDITY OF CALCULATING THE CYTOPLASMIC FREE [NAD+]/[NADH] [H+] RATIO FROM SUBSTRATE CONCENTRATIONS OF BRAIN

Abstract— This study is an investigation into the applicability of redox calculations to brain. At six intervals following electroconvulsive scizure, multiple metabolites were measured in freeze-blown brain from unanesthetized rats. From substrate ratios, the time course of the rapid changes in the cytoplasmic free [NAD⁺]/ [NADH] [H⁺] ratio was calculated from the reactions of lactate dehydrogenase [EC 1.1.1.27], malate dehydrogenase [EC 1.1.1.37] and glycerolphosphate dehydrogenase [EC 1.1.1.8], The pattern of the redox ratios in the control animals was also compared with the same ratios determined in freeze-clamped liver, a relatively homogeneous tissue. Though some evidence for effects of compartmentation are present in the results from brain, these effects are relatively minor. There was found to be very good agreement in the direction and magnitude of change of the redox ratios calculated from lactate dehydrogenase and malate dehydrogenase, and even from glycerolphosphate dehydrogenase at points of low flux. In spite of rapid changes of metabolites, the reaction catalyzed by glutamateoxaloacetate transaminase remained very near its equilibrium position at all time periods. From the results it has been concluded that in spite of the obvious structural heterogeneity of brain, meaningful calculations of the cytoplamic redox state in brain are possible.     

Allosteric Properties of Ox Brain Nicotinamide–Adenine Dinucleotide Dependent Isocitrate Dehydrogenase

Abstract: The kinetic and regulatory properties of a partly purified preparation of ox brain NAD⁺-dependent isocitrate dehydrogenase have been studied at pH 7.5. The enzyme exhibits rate cooperativity with respect to isocitrate but shows normal hyperbolic kinetics with respect to NAD⁺. ADP activates the enzyme by decreasing the substrate concentrations that are necessary to give half-maximal velocity, but it has no effect on the Hill constant for isocitrate unless Mg²⁺ ions are replaced by Mn²⁺ ions in the reaction mixture. Citrate and tricarballylate activate the enzyme in a similar fashion to ADP. Higher concentrations of citrate cause inhibition but this could be overcome by raising the concentration of Mg²⁺ ions, suggesting that the inhibition by this compound might be due to its acting as a chelating agent. NADH and NADPH were competitive inhibitors with respect to NAD⁺ but the product, 2-oxoglutarate, was not inhibitory. γ-Aminobutyrate and a number of other compounds involved in the γ-aminobutyrate pathway had no significant effect on the activity of the enzyme.     

NADP+-malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme

The thioredoxin-dependent light/dark modulation system of the chloroplast is described as a prerequisite enabling the flexible control of fluxes through the various parts of the CO₂-fixation pathway. Both the rapid turnover of the reduced thiol-containing form of the respective target enzyme, and the metabolite effect upon the reductive enzyme modulation, allow rapid adjustment of the amount of active species to the actual requirements. The structural basis of the regulation of chloroplast NADP⁺-malate dehydrogenase (EC 1.1.1.82) is described in more detail. The modulable plastid enzyme is characterized by two sequence extensions not present in any other known NADP⁺- and/or NAD⁺-specific malate dehydrogenase. The NADP⁺-malate dehydrogenase of C₃-plants is part of the "malate valve", which catalyzes the export of reducing equivalents in the form of malate from the chloroplast only when the NADPH to NADP⁺ ratio is high, thus poising the NADPH to ATP ratio required for optimal carbon reduction in the light. The mode of regulation of other light/dark modulated enzymes is discussed.     

A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K⁺]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD⁺/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.     

The seven NAD(H)-glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities

The specific activities of aminating NADH- and deaminating NAD⁺-glutamate dehydrogenase (GDH, EC 1.4.1.2) varied considerably in crude extracts of grapevine ( Vitis vinifera L. cv. Sultanina) callus and were dependent on the nitrogen source of the culture medium. However, dialysis of the enzyme preparations resulted in a significant decrease in the deaminating GDH specific activity while the aminating activity was not affected. The presence of malate in the crude extract resulted in erroneous overestimation of the NAD⁺-GDH activity through the malate dehydrogenase reaction. Thus, in dialysed extracts, the ratio of the NADH-GDH/NAD⁺-GDH specific activities remained relatively constant irrespective of the nitrogen source. In view of this evidence, we now have modified methods for staining both the NADH-GDH and NAD⁺-GDH activities on gels in order to compare the aminating and deaminating activities of each of the 7 GDH isoenzymes. The results from the staining of NADH-GDH and NAD⁺-GDH activity of enzyme preparations from calluses revealed the same isoenzyme profile. Furthermore, separated leaf isoenzymes showed similar activity ratios and kinetic properties. These results may suggest that each one of the 7 isoenzymes have similar in vitro anabolic and catabolic activities.     

The role of acetaldehyde and glycerol in the adaptation to ethanol stress of Saccharomyces cerevisiae and other yeasts

Ethanol inhibition is a commonly encountered stress condition during typical yeast fermentations and often results in reduced fermentation rates and production yields. While past studies have shown that acetaldehyde addition has a significant ameliorating effect on the growth of ethanol-stressed Saccharomyces cerevisiae , this study investigated the potential ameliorating effect of acetaldehyde on a wide range of ethanol-stressed yeasts. Acetaldehyde does not appear to be a universal ameliorating agent for yeasts exposed to ethanol stress. It is also shown that as a result of an ethanol stress, most yeasts rapidly produce glycerol as an alternative means of NAD⁺ regeneration rather than having a specific requirement for glycerol. The results strongly suggest that both ethanol and acetaldehyde exposure have a direct effect on the cellular NAD⁺/NADH ratio, which can manifest itself as modulations in glycerol production.     

FREEZE-BLOWING: A NEW TECHNIQUE FOR THE STUDY OF BRAIN IN VIVO

Abstract— A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N₂. This method stops brain tissue metabolism more rapidly than the previously-described methods of microwave irradiation, decapitation into liquid N₂, or whole-animal immersion into liquid N₂, as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze-blown brain had higher levels of a-oxoglutarate, creatine phosphate, pyruvate, glucose and glucose-6-phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD⁺]/[NADH₂], [NADP⁺]/[NADPH₂] and [ATP]/[ADP] [HPO₄^2-] ratios were higher in freeze-blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze-blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze-blowing technique more closely resemble those which occur in vivo.     

Properties of NADP+-malic enzyme from pod walls of chickpea (Cicer arietinum)

NADP⁺-malic enzyme ( l -malate: NADP⁺ oxidoreductase, decarboxylating EC 1.1.1.40) from pod walls of chickpea was purified 51-fold by ammonium sulphate fractionation, DEAE- cellulose chromatography and gel filtration through Sepharose 4B. The purified enzyme required a divalent cation, either Mn²⁺ or Mg²⁺, for its activity. K_m values at pH 7.8 for malate, NADP⁺ and Mn²⁺ were 4.0, 0.031 and 0.71 m M , respectively. Mn²⁺-dependent activity was inhibited by heavy metal ions such as Cd²⁺, Zn²⁺, Hg²⁺, and to a lesser extent by Pb²⁺ and Al³⁺. Among the organic acids examined, sodium salts of oxalate and oxaloacetate were inhibitory. Kinetics of the reaction mechanism showed sequential binding of malate and NADP⁺ to the enzyme. Products of reaction, viz. pyruvate, bicarbonate and NADPH, inhibited the enzyme activity. At limiting concentrations of NADP⁺, pyruvate and bicarbonate induced a positive cooperative effect by malate. It is proposed that the activity of NADP⁺-malic enzyme is controlled by intracellular concentrations of substrates and products.     

4-Aminobutyraldehyde Dehydrogenase Activity in Rat Brain

Abstract: An enzyme with NAD⁺-dependent 4-aminobutyraldehyde dehydrogenase activity was purified about 360-fold from rat brain extract. AMP-Sepharose chromatography was effective in separating the enzyme from other NAD⁺-dependent aldehyde dehydrogenases included in the extract. The K _ms for the substrates NAD⁺ and 4-aminobutyraldehyde were 4.8 × 10⁻⁴ and 8.3 × 10⁻⁵ M , respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4-aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4-aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.