Nicotinamide Adenine Dinucleotide Phosphate Phosphatase Facilitates Dark Reduction of Nitrate: Regulation by Nitrate and Ammonia

Leaves of 15 - 30-d-old plants of sunflower and jute were harvested at 10.00 or 23.00 (local time) and measured immediately, or those harvested at 10.00 were incubated for one hour in sunlight either in water or 5 mM methionine sulfoximine (MSX) solution and then for three hours in dark either in water or 15 mM KNO3 solution. Nitrate feeding during dark incubation, in general, increased nitrate reductase (NR) and nitrite reductase (NiR) activities, and NADH and soluble sugar contents. Increase in tissue nitrate concentration in MSX fed but not in control samples suggested reduction of nitrate in dark. NADPH-dependent NR activity increased considerably upon feeding with nitrate in dark. Concomitantly, NADPH phosphatase activity was also increased in nitrate treated, dark incubated leaves. It is proposed that nitrate regulates dark nitrate reduction by facilitating generation of NADH from NADPH by NADPH phosphatase. High amounts of ammonia accumulated in MSX treated, but not in control leaves, upon dark incubation. Relative activities of NR and NADPH phosphatase, and amounts of soluble sugar and NADH were low in MSX fed samples compared to that of control. So, high amount of ammonia might partially repress NADPH phosphatase and consequently deprive NR of reducing equivalents. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Distribution of Enzymes Which Synthesize Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate in Monkey, Rabbit, and Ground Squirrel Retinas

The activity of adenosinetriphosphate:nicotinamide adenylyltransferase (EC 2.7.7.1) was measured in all the layers of monkey, rabbit, and ground squirrel retinas. Nicotinamide adenine dinucleotide (NAD) kinase (EC 2.7.1.23) distribution was measured in monkey and rabbit retinas. An attempt was made to measure NAD synthetase (EC 6.3.5.1), but the activities in the retinal layers were too low to produce a reliable increment in the levels of endogenous NAD. In monkey retina the adenylyl transferase was highest by far in the outer and inner nuclear layers, lower and variable in ganglion cell and fiber layers, and almost absent elsewhere. Rabbit retina differed in that activity was nearly absent in the outer nuclear layer, whereas in the ground squirrel outer nuclear layer activity was double that of the inner nuclear layer. The species differences suggest that adenylyl transferase is almost absent from cone cell nuclei and high in rod cell nuclei. NAD kinase distribution in monkey retina was almost the mirror image of that of adenylyl transferase.     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Production of Nicotinamide Adenine Dinucleotide Glycohydrolases by Bacteria

The distribution of heavy metals (Fe, Mn, Zn, Cu, Pb, Ni and Cd) were investigated in various organs and tissues of striped dolphin, Stenella coeruleoalba. The animals were caught alive at Taiji, on the coast of Kii Peninsula, during the open season in December 1978. Determination of the metals was made by atomic absorption spectrophotometry and significant differences of metal concentration in the positions of the muscle, blubber and skin, respectively, were observed. The front ventral muscle of matured dolphins showed the highest concentrations of Zn and Cd and lowest Fe when compared to other parts of the muscle. Most of the metals recorded relatively low concentrations in melon rather than in the other lipid layers of blubber. In skin tissue, the concentrations of Fe, Mn and Zn were significantly higher in black-colored skin than in white skin. Moreover, a difference in the concentrations of metals according to bone position was observed. In general, high concentrations of most of the metals were found in liver, kidney and bone, with low concentrations in brain and the lipid layer of blubber. Furthermore, relatively high concentrations of Cu, Mn and Zn were found in skin, and for Mn, Zn, Ni and Cd it was likewise in pancreas and the reproductive organs. Based upon these results, the nature of the organ(s) of a dolphin that has to be selected for ecological and hygienic comparison was discussed.     

Photosynthetic Activities of the Halophilic Alga Dunaliella parva

Dunaliella parva, a unicellular halophilic alga, was found to evolve oxygen photosynthetically only in the presence of a high osmolar concentration. Cell free preparations were obtained by placing the cells in a medium of low osmolarity. The fragments obtained showed a high photoreducing and photophosphorylating activity except for their inability to catalyze all ferredoxin dependent photoreactions. Placing the cells in a medium of intermediate osmolarity produced a “chloroplast” preparation which maintained some capacity for O₂ evolution and CO₂ fixation, while possessing the ability to catalyze the photoinduced reduction of ferricyanide. Enzymic and photosynthetic reactions of cell-free preparations from D. parva were inhibited, rather than stimulated, by the salt concentration optimal for growth. These results were interpreted as indicating the existence of a steep NaCl gradient in vivo between the medium and the cell compartments which are not permeable to salt.     

Function of Ubiquinone in Electron Transport from Reduced Nicotinamide Adenine Dinucleotide to Nitrate and Oxygen in Aerobacter aerogenes

The possible role of quinones in the electron transport system of Aerobacter aerogenes was investigated. The only quinone found in measurable amounts in bacteria grown in minimal media under both aerobic and anaerobic conditions was ubiquinone-8. Membrane-bound ubiquinone-8 could be removed by extraction with pentane, or destroyed by ultraviolet irradiation, with a concomitant loss of both reduced nicotinamide adenine dinucleotide (NADH) oxidase and NADH-linked respiratory nitrate reductase activity. In the extracted membrane preparations, these enzymatic activities could be restored, both to the same degree, by incorporation of ubiquinone-6, -8, or -10, but not by incorporation of menaquinones. The NADH oxidation and the nitrate reduction were sensitive to the respiratory inhibitors dicoumarol, lapachol, and cyanide. The results obtained indicate that ubiquinone-8 mediates the electron transport between NADH and oxygen as well as between NADH and nitrate. Branching of the electron transport chain to oxygen and nitrate occurs after an initial common pathway.     

Properties of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum

Li, Lan-Fun (Western Reserve University School of Medicine, Cleveland, Ohio), Lars Ljungdahl, and Harland G. Wood. Properties of nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase from Clostridium thermoaceticum. J. Bacteriol. 92: 405-412. 1966.-A nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase has been isolated from C. thermoaceticum. The enzyme is very sensitive to oxygen and requires sulfhydryl compounds for activity. The apparent K(m) at 50 C and pH 7.0 for NADP is 5.9 x 10(-5)m and for formate, 2.2 x 10(-4)m. The enzyme is most active at about 60 C and at pH values between 7.0 and 9.0. The enzyme catalyzes an exchange between C(14)O(2) and formate, which requires NADP, but net synthesis of formate from CO(2) and reduced nicotinamide adenine dinucleotide phosphate could not be demonstrated. The reaction does not involve ferredoxin.     

Production of Nicotinamide Adenine Dinucleotide by Saccharomyces carlsbergensis

The accumulation of NAD was studied by culturing yeast in the presence of NAD precursors, Among the strains tested, Saccharomyces carlsbergensis showed the highest ability for the accumulation of NAD, Additions of pantothenate, inositol, zinc ion and fatty acids were effective for the accumulation of NAD. Under the optimal culture condition, NAD level in Saccharomyces carlsbergensis reached 42 mg per gram dry cells. Surfactants belonging to alkyl sulfate were effective on the leaking of the intracellular NAD, and about 75 mg of NAD per 100 ml was accumulated.     

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane （PM） nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content （RWC） and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H2O2 and O2^- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants. Histochemical analyses also revealed increased H2O2 and O2^- production in drought-stressed leaves. Application of diphenylene iodonium （DPI）, an inhibitor of PM NADPH oxidase, did not alleviate drought-induced production of H2O2 and O2^-. Catalysis experiments indicated that the rice PM NADPH oxidase was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H2O2 system, is involved in the response of rice to drought stress.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

The Reduction of Nicotinamide Adenine Dinucleotide by Mitochondria Isolated from Helianthus tuberosus

Intact mitochondria isolated from Jerusalem artichoke tubersoxidize both malate and citrate. Both substrates were equallyeffective in reducing the endogenous pool of pyridine nucleotidesin the presence of respiratory inhibitors. Only malate, andnot citrate, was capable of reducing exogenous NAD⁺ under similarassay conditions. The rate at which malate reduced added NAD⁺was biphasic; the initial rapid phase was inhibited by the accumulationof oxaloacetate while the velocity of the second, slower, phasewas found to be insensitive to accumulated oxaloacetate. Theaddition of the detergent Decon 90 to intact mitochondria stimulatedboth the rapid and slow phases of NAD⁺ reduction and failedto convert the biphasic rate into a constant rate. Decon 90was found to cause inhibition of soluble malate dehydrogenase.Extensive efforts to purify the mitochondria using sucrose densitygradients failed to remove all of the soluble malate dehydrogenasefrom the preparation of mitochondria and approximately 3% ofthe total malate dehyrogenase present in the preparation appearedto behave as if it were outside the inner membrane.     

The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondria

The oxidation of malate, citrate, and α-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD⁺ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD⁺ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD⁺.     

Attempts to Detect an Alternative Vital Role for the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase Structural Gene in Aspergillus nidulans

A possible alternate vital role of the structural gene for reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase in Aspergillus nidulans was tested for and found to be absent in a significant number of haploid and diploid strains.     

Photoregulation of Nicotinamide Adenine Dinucleotide Kinase Activity in Cell-free Extracts

This article gives evidence that NAD kinase activity is controlled by the action of phytochrome. The NADP level rapidly increased in the cotyledons of seedlings of Pharbitis nil strain Violet (a short day plant), when the inductive dark for flowering was interrupted with a 5-minute illumination of red light. Illumination with far red light immediately after illumination with red light counteracted partly the effect of the latter.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Salt-dependent Protein Composition in the Halotolerant Green Alga,Dunaliella parva

The extremely halotolerant green alga, Dunaliella parva, tolerates salt concentrations from 0.3 to 3.0 M NaCl. Effects of long-term adaptation to five distinct salinities were analyzed. Salt-dependent differences of physiological parameters such as growth rate, pigments, quantitative protein contents, and gas exchange were measured; furthermore the qualitative protein composition in salt-adapted cells was investigated using SDS-polyacrylamide gel electrophoresis. Proteins of apparent molecular masses of 26, 35, 39, 50, and 63 kDa were induced or intensified with an increase in external sodium chloride concentration whereas proteins of 85 and 101 kDa were diminished in high salt algae. After selective staining, four modifications of glycoproteins were observed. A glycoprotein of 96 kDa was produced exclusively in low salt cells whereas glycosylations of 105, 135, and 260 kDa were induced by high salt concentrations.     

Kinetics of Activation of Nicotinamide Adenine Dinucleotide Kinase by Phytochrome-Far Red-absorbing Form

The effects of exogenous redox cofactors and purine analogues on the activation of the NAD kinase by Pfr were examined. Addition of phenazine methosulfate, flavin mononucleotide, or methylene blue increased the activation of NAD kinase by red light in a partially purified preparation of phytochrome. Phenazine methosulfate and flavin mononucleotide do not absorb light in the red or far red region, so they do not act as light receptors in this activation. Thus they probably intervene in electron transfer between phytochrome and NAD kinase. Addition of kinetin with these compounds increased photopotentiation further. In the presence of phenazine methosulfate and kinetin, the activation of NAD kinase by red light was counteracted by illumination with far red light immediately after red light.     

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta

Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants.