The responses of isolated plant mitochondria to external nicotinamide adenine dinucleotide

The effects of added NAD on substrate oxidation by turnip (Brassica rapa L.) and beetroot (Beta vulgaris L.) mitochondria were investigated. State 3 malate and 2-oxoglutarate oxidation rates with turnip mitochondria were stimulated 25 to 40% by external NAD. Following NAD-depletion this stimulation by NAD was increased to 70 to 80%. With purified beetroot mitochondria, state 3 malate and 2-oxoglutarate oxidation rates were only marginally increased (10-15%) by the addition of NAD but after NAD-depletion treatments this stimulation increased to 55%. The effect of added NAD on oxidation rates could be reduced by preloading mitochondria with NAD in the presence of succinate. Oxidation rates were found to be most sensitive to the addition of external NAD when rotenone was present. The uptake of external NAD into beetroot mitochondria appeared to be composed of both an active and a diffusive component. The active component displayed saturation kinetics with an approximate K_m of 0.105 ± 0.046 millimolar. These results provide further evidence, reported previously with potato mitochondria, that NAD can move across the inner membrane of plant mitochondria. They are particularly significant with respect to beetroot mitochondria which in contrast to other plant mitochondria, have not demonstrated any response to added NAD.     

Factors affecting the redox state of bovine epididymal spermatozoa.

The oxidation-reduction state of bovine epididymal spermatozoa was determined in vitro by fluorescence spectroscopy and by direct chemical analysis. Enhanced NADH fluorescence in sperm was observed with the onset of anaerobiosis in the sample cuvette. However, part of this increased fluorescence was temporary and a stable pyridine nucleotide fluorescence was not reached until 25 min after the onset of anaerobiosis. The transient was not paralleled by an equivalent increase in cellular NADH as measured by absorption spectroscopy. Hypotonic treatment of sperm, which removed the plasma membrane, liberated greater than 50% of the cellular NAD and that remaining was reduced by rotenone addition, indicating its mitochondrial location. Hypotonically treated sperm did not demonstrate a transient fluorescence above that due to the increases in NADH from anaerobiosis. Addition of pyruvate to anaerobic sperm resulted in a rapid decrease in fluorescence that corresponded to NADH oxidation coupled with the reduction of pyruvate to lactate. The duration of this oxidized state was dependent on the amount of pyruvate added. Analysis of cellular NAD under similar conditions confirmed this result. The pyridine nucleotides of hypotonically treated cells were also oxidized by pyruvate but were not reduced by added glucose as in untreated sperm. These results indicate that pyruvate reduction served to balance reducing equivalents and temporarily reoxidized the intracellular milieu of the anaerobic spermatozoon. The data also support the hypothesis that pyruvate and lactate can serve as reducing equivalent carriers between cytosol and mitochondria.     

Unconventional and effective methods for the regeneration of NAD(P) H in microorganisms or crude extracts of cells

Several yeasts, as well as aerobic and anaerobic bacteria catalyze the reduction of NAD and NADP in the presence of reduced methylviologen. The rates are usually much higher than those of reductions of unsaturated substrates by the organisms in cofermentations with carbohydrates. Since methylviologen can be continuously reduced at the cathode of an electrochemical cell it acts in catalytic amounts as a regenerable electron donor. Such systems may be superior to that with glucose as electron donor, because the NAD(P)H can be used exclusively for the reduction of the unsaturated substrate. The rate of the NAD(P)H formation depends very much on the organism and for the same organism on the growth procedure, the growth medium, the pretreatment of the cells, the pH, the buffer as well as on the ionic strength. Cells of Candida utilis which were frozen and thawed several times were superior to cells freshly harvested. Crude extracts revealed the best activities.Clostridia show the highest activities (up to 15 U per mg protein in the crude extract) and are suitable catalysts for the preparation of [4S-²H]NADH and [4S-²H]NADPH using ²H₂O-buffer in an electrochemical cell.The combinations of Alcaligenes eutrophus or Clostridium kluyveri and Candida utilis extracts in the presence of methylviologen are effective systems to reduce hydroxyacetone with hydrogen gas as electron donor or in an electrochemical cell. In this combination of microorganisms NADH is formed mainly by A. eutrophus or C. kluyveri and consumed for the reduction of hydroxyacetone by a reductase present in Candida utilis. The productivity numbers of such combinations are 10–30 times higher than those of yeasts alone.NAD(P)H regeneration, methylviologen-dependent NAD(P)H formation, deuterated NAD(P)H, Clostridia, yeast, bioreduction     

Energy metabolism in Saccharomyces cerevisiae. Effect of progressive starvation on respiration and pyridine nucleotide reduction linked to ethanol oxidation in intact cells

The progressive effects of aerobic starvation on endogenous and ethanol-linked respiration and pyridine nucleotide reduction have been studied in the yeast Saccharomyces cerevisiae. Three distinct phases of pyridine nucleotide reduction were observed when ethanol was added to unstarved yeast: an initial phase of rapid reduction and accelerating respiration (A); a steady-state phase of reduction with maximal respiration (B); a final phase of rapid reduction at anaerobiosis (C).During the first 5 hr of aeration, the steady-state Phase B was replaced by a phase of slow pyridine nucleotide reduction, while Phases A and C were unaffected. During this period, both endogenous pyridine nucleotide reduction and endogenous respiration decreased sharply.Between 5 and 22 hr of aeration, the endogenous level of reduced pyridine nucleotide declined further, while endogenous respiration remained unchanged. Concurrently, the extent of the Phase A reduction doubled.The addition of ethanol to aerobic, unstarved yeast stimulated a rapid pyridine nucleotide reduction, with further reduction occurring at anaerobiosis. Under anaerobic conditions, the addition of ethanol to unstarved yeast caused little further reduction of pyridine nucleotide. Two hours of starvation decreased the extent of the endogenously supported anaerobic reduction and correspondingly increased the ethanol-induced reduction. These results suggest that, in unstarved yeast, reducing equivalents derived from ethanol under aerobic conditions and those derived from endogenous carbohydrate under anaerobic conditions have access to the same pool of pyridine nucleotide. With starvation, this pool becomes accessible to ethanol-derived (or ethanol-mobilized) reducing equivalents under anaerobic conditions.     

The Kinetics of Pyridine Nucleotide Reduction following the Addition of Glucose to Bakers' Yeast

Changes of pyridine nucleotide concentration in yeast followingthe addition of glucose have been measured by a fluorimetricassay applied to cell extracts. The major changes were in nicotinamideadenine dinucleotide (NAD) which, during the first minute afterglucose addition, was rapidly reduced and then more slowly reoxidized.Later, one to two minutes after adding glucose, NAD was reducedagain to a steady-state value. No NAD was reduced when acetaldehydewas added simultaneously with glucose. The oxidation of NAD,which followed the initial reduction, was slowed down by sodiumfluoride and almost completely abolished by sodium sulphite.The initial reduction of NAD is ascribed to 3-phosphoglyceraldehydedehydrogenase and its subsequent oxidation is ascribed to alcoholdehydrogenase. Probably only that NAD bound to 3-phosphoglyceraldehydedehydrogenase is reduced initially. The reasons for these viewsare discussed.     

Regulation of cyclic photophosphorylation in Rhodospirillum rubrum by the redox state of nicotinamide-adenine dinucleotide

We have investigated the effect of the redox state of added NAD on the rates of anaerobic cyclic photophosphorylation which are supported by membrane vesicles isolated from Rhodospirillum rubrum. As the redox potential of NAD was lowered, the activity decreased according to a typical potentiometric titration. The Nernst plot showed an apparent midpoint potential (E′_o) of ?350 mV and had a slope which corresponded to a two-electron transition. Besides, an almost identical potentiometric relationship was found to exist between the extent of light-elicited ATP formation in anaerobic suspensions of intact R. rubrum cells and the redox potential of intracellular NAD. These results suggest that physiological photophosphorylation in R. rubrum requires the oxidized form of a membrane-bound constituent (E′_o = ?350 mV) whose redox state is controlled by the redox state of cytoplasmic NAD.     

Depletion of casein kinase I leads to a NAD(P)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis

Background

In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag⁻ phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing.

Methods

A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses.

Results

Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced.

Conclusions

The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)⁺/NAD(P)H redox balance state.

General significance

The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.     

Utilization of Saccharomyces cerevisiae recombinant strain incapable of both ethanol and glycerol biosynthesis for anaerobic bioproduction

The yeast Saccharomyces cerevisiae produces ethanol and glycerol as major unwanted byproducts, unless ethanol and glycerol are the target compounds. Minimizing the levels of these byproducts is important for bioproduction processes using yeast cells. In this study, we constructed a yeast strain in which both ethanol and glycerol production pathways were disrupted and examined its culture characteristics. In wild-type yeast strain, metabolic pathways that produce ethanol and glycerol play an important role in reoxidizing nicotinamide adenine dinucleotide (NADH) generated during glycolysis, particularly under anaerobic conditions. Strains in which both pathways were disrupted therefore failed to grow and consume glucose under anaerobic conditions. Introduction of desired metabolic reaction(s) coupled with NADH oxidation enabled the engineered strain to consume substrate and produce target compound(s). Here we introduced NADH-oxidization-coupled L-lactate production mechanisms into a yeast strain incapable of ethanol and glycerol biosynthesis, based on in silico simulation using a genome-scale metabolic model of S. cerevisiae. From the results of in silico simulation based on flux balance analysis, a feasible anaerobic non-growing metabolic state, in which L-lactate yield approached the theoretical maximum, was identified and this phenomenon was verified experimentally. The yeast strain incapable of both ethanol and glycerol biosynthesis is a potentially valuable host for bioproduction coupled with NADH oxidation under anaerobic conditions.     

The estimation of alcohol dehydrogenase activity in aerobic and anaerobic “permeabilised” baker’s yeast cells

The in vivo and in vitro activity of alcohol dehydrogenase from baker's yeast maintained under aerobic and anaerobic conditions was measured. In vivo measurements were made in cells "permeabilised" with toluene. Michaelis constants (NAD+ as substrate) were found to be almost identical as those reported for purified preparations. In addition the Km of the enzyme from cells incubated under anaerobic conditions was virtually identical to that from cells from aerobic conditions. The activity of the enzyme was found to be greater (in both "permeabilised" cells and extracts) in cells maintained under nitrogen than air. Cells metabolizing glucose in N2 produced greater levels of ethanol than in air and the rate of NAD+ reduction was also found to be greater in N2 than in air. The results indicate that it was feasible to determine rates of this enzyme in vivo and that the difference in activity of alcohol dehydrogenase under N2 and air may conceivably account for differences in rates of glucose utilisation, ethanol production and NAD+ reduction in air and nitrogen.     

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts

Chlorophyll a fluorescence rise kinetics (from 50 μs to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O₂-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells cultivated aerobically or anaerobically

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells were examined on the basis of the cell growth rate, NAD(P)H concentration, reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. In aerobically or anaerobically cultured yeast cells, NAD(P)H concentration decreased with increasing concentration of menadione, and the recovery of NAD(P)H concentration was proportional to the cell growth rate. However, there was no relationship among the inhibition of the cell growth and reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. Among them, ethanol production showed resistance to the cytotoxicity of menadione, suggesting the resistance of glycolysis to menadione. The growth inhibitory effect of menadione depended on the rapid decrease and the recovery of NAD(P)H rather than production of reactive oxygen species regardless of aerobic culture or anaerobic culture and presence or absence of mitochondrial function. The recovery of NAD(P)H concentration after the addition of menadione might depend on menadione-resistant glycolytic enzymes.     

Occurrence of cytochromes in R and S yeast mutants of the genusSaccharomyces

Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a₃ band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b₁ was connected with their inability to reproduce under anaerobic conditions.     

Activities of isolated mitochondria and mitochondrial enzymes from aerobically and anaerobically germinated barnyard grass (echinochloa) seedlings

Activity of mitochondria isolated from whole seedlings of Echinochloa crus-galli (L.) Beauv. var oryzicola germinated under aerobic and anaerobic conditions for 5 to 7 days was investigated. Mitochondria from both treatments exhibited good respiratory control and ADP/O ratios. Although O₂ uptake was low in anaerobic mitochondria, activity rapidly increased when the seedlings were transferred to air. Mitochondria from both aerobically and anaerobically grown seedlings of E. crus-galli var oryzicola maintained up to 66% of their initial respiration rate in the presence of both cyanide and salicylhydroxamic acid, and the inhibitory effects of cyanide and azide were additive. In addition, antimycin A was not an effective inhibitor of respiration. Reduced-minus-oxidized absorption spectra revealed that cytochromes a, a₃, and b were reduced to a greater extent and cytochrome c was reduced to a lesser extent in anaerobically germinated seedlings relative to that in aerobically germinated seedlings. An absorption maximum in the cytochrome d region of the spectrum was reduced to the same extent under both germination conditions and an absorption maximum at 577 nm was present only in anaerobically germinated seedlings. Anaerobically germinated seedlings contained 70% of the cytochrome c oxidase activity found in air grown seedlings. Upon exposure to air, the developmental pattern of this enzyme in anaerobically germinated seedlings was similar to air controls. Succinate dehydrogenase activity in anaerobic seedlings was only 45% of the activity found in aerobically germinated seeds, but within 1 hour of exposure to air, the activity had increased to control levels. The results suggest that mitochondria isolated from E. crus-galli var oryzicola differ from other plants studied and that the potential for mitochondrial function during anaerobiosis exists.     

Oxygen Requirements of the Food Spoilage Yeast Zygosaccharomyces bailii in Synthetic and Complex Media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Der Einfluß der Kulturbedingungen auf den Nicotinamid-Adenin-Dinucleotid(phosphat)-Gehalt in Zellen von Rhodospirillum rubrum

Zusammenfassung In Zellen von R. rubrum war das Verhältnis von oxydiertem zu reduziertem NAD(P) vom Sauerstoffpartialdruck im Medium, der Lichtintensität und der Nährbodenzusammensetzung abhängig. In ruhenden Kulturen unter aeroben Bedingungen im Licht oder im Dunkeln und anaerob bei hoher Lichtintensität, wenn der ATP-Pool in den Zellen groß ist, beobachtete man einen relativ hohen Wert für das Verhältnis von NAD(P)⁺/NAD(P)H. Unter Kulturbedingungen, bei denen der ATP-Gewinn der Zellen gering ist (anaerob Schwachlicht oder anaerob Dunkel), sank das Verhältnis von NAD(P)⁺/NAD(P)H ab. Die niedrigsten Werte für das Verhältnis von NAD(P)⁺/NAD(P)H wurden dementsprechend in anaerober Dunkelkultur, die höchsten in aerober Lichtkultur gefunden.Anaerob im Dunkeln war der NAD(P)H-Spiegel auch vom Substrat abhängig: mit Fructose oder ohne Substrat beobachtete man einen sehr großen NAD(P)H-Pool in den Zellen; nach Zugabe von Acetat, Succinat, Pyruvat oder Malat sank der Spiegel der reduzierten Coenzyme ab.In wachsenden Kulturen (außer anaerob im Dunkeln) nahm die relative Konzentration von NAD⁺ und der NADP⁺-Pool im Vergleich zu ruhenden Zellen stark zu (3-5fach).Änderungen im Verhältnis von NAD⁺/NADH und von NADP⁺/NADPH waren aber nicht unter allen Kulturbedingungen direkt korreliert.Es wird diskutiert, wieweit das Adenylatsystem und das NAD(P)-System einen regulativen Einfluß auf die Bacteriochlorophyll-Synthese und die Morphogenese bei Athiorhodaceae haben.

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The influence of culture conditions on the NAD(P) content of Rhodospirillum rubrum cells

Summary In cells of R. rubrum the ratio of oxidized to reduced NAD(P) depended on the oxygen pressure in the medium, the light intensity, and the composition of the medium. The ratio of NAD(P)⁺/NAD(P)H was high under conditions when the ATP-pool in the cell is large, viz. in resting cultures either kept aerobically in the light or in the dark or kept anaerobically in strong light. The quotient NAD(P)⁺/NAD(P)H decreased under conditions of reduced ATP-synthesis in the cells (anaerobic in dimlight or in the dark). Consequently, the lowest NAD(P)⁺/NAD(P)H value was observed in anaerobic dark cultures, the highest in aerobic light cultures.Under anaerobic conditions in the dark, the NAD(P)H level depended also on the substrate: with fructose or without any substrate, a large NAD(P)H pool was observed; the level of reduced coenzymes decreased upon addition of acetate, succinate, pyruvate, or malate.In growing cultures (except under anaerobic conditions in the dark) the relative concentration of NAD⁺ and the NADP⁺ pool showed a considerable increase (3 to 5 fold), as compared with resting cells. However, the changes in the proportions of NAD⁺/NADH and NADP⁺/NADPH were not directly correlated under all culture conditions.The regulative influence of the adenylate and the NAD(P) systems on the synthesis of bacteriochlorophyll and morphogenesis in Athiorhodaceae is discussed.

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Abkürzungen BChl Bacteriochlorophyll a - NAD(P) NAD-Nucleotide=reduziertes und oxydiertes Nicotinamid-Adenin-Dinucleotid und Nicotinamid-Adenin-Dinucleotidphosphat Herrn Prof. Dr. H. Engel zum 70. Geburtstag gewidmet.     

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.     

Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica

NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6–2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.     

Variation of nicotinamide adenine dinucleotide phosphate level in bean hypocotyls in relation to o(2) concentration

Slices of hypocotyls from 3-day-old seedlings of Vigna sesquipedalis (L.) Fruwirth in the germination stage were incubated under various gaseous conditions. The NADP+NADPH level in the hypocotyl slices changed with the oxygen tension. A high NADP+NADPH level was observed under aerobic conditions and a low NADP+NADPH level under anaerobic conditions.

The 100 × NADH/NAD+NADH ratio increased greatly under anaerobic conditions. In general a low NADP + NADPH level corresponded with a high 100 × NADH/NAD+NADH ratio. On the basis of the results given in the following paper, it was discussed that the slowness of NADH oxidation in hypocotyl tissue due to anaerobic conditions results in the inhibition of NADP formation.

The variation of the NADP+NADPH level was considered to produce a modification of the carbohydrate metabolism.

The NADP+NADPH level in E. coli cells suspended in glucose solution also changed with the oxygen tension.

     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

Effect of aeration during cell growth on ketone reactions by immobilized yeast

The effect of aeration during cell growth on the subsequent reduction of 2-hexanone and 2-octanone by yeast cells entrapped in calcium alginate beads was studied. The reactions were conducted using 2-propanol as a sacrificial substrate to regenerate the cofactor NAD(H), and a mixture of (S)- and (R)-alcohols was produced. The use of strictly aerobic conditions when growing the cells resulted in the highest initial reaction rates, as well as the production of only a single product (i.e., the enantiomeric excess of the (S)-alcohols was 1.0). However, initial reaction rates decreased proportionally with fermentation time regardless of whether the yeast were grown aerobically or under both aerobic and anaerobic conditions. The data also suggest that it is the aerobic (or anaerobic) condition, rather than the cell growth phase, which is responsible for the results seen.