Levels of Nicotinamide Adenine Dinucleotide and Reduced Nicotinamide Adenine Dinucleotide in Facultative Bacteria and the Effect of Oxygen

Nicotinamide adenine dinucleotide (NAD) and reduced NAD (NADH) levels have been measured in bacterial cultures. The cofactors were assayed by using the very sensitive cycling assay described previously by Cartier. Control experiments showed that the level of total NAD(H) falls during harvesting, and so samples were taken quickly from growing cultures and extracted immediately without separating the cells from the medium. Total NAD(H) ranged from 4.0 to 11.7 mumoles/g of dry cells for three facultative organisms, Klebsiella aerogenes, Escherichia coli, and Staphylococcus albus. NADH was remarkably constant in these bacteria; only one out of ten series of determinations was outside the range 1.4 to 1.9 mumoles/g of dry cells. NAD(+) showed much greater variation. An anaerobe (Clostridium welchii) had significantly more total NAD(H) whereas an aerobe Pseudomonas aeruginosa had about as much NAD(H) as the facultative organisms. NAD and NADH were measured during growth: once more NADH was much more constant than NAD. During change-over between aerobiosis and anaerobiosis, NADH showed a temporary increase but then returned to a constant level, whereas NAD changed from high aerobically to low anaerobically. These results are discussed in terms of the control mechanisms that may be involved.     

Phosphorylation and the Reduced Nicotinamide Adenine Dinucleotide Oxidase Reaction in Streptococcus agalactiae

Cell-free extracts from aerobically grown Streptococcus agalactiae cells possess a reduced nicotinamide adenine dinucleotide (NADH) oxidase which is linked to oxygen. It is inhibited by cyanide, although cytochromes evidently are not involved. Adenosine triphosphate (ATP) formation occurs during the reaction, but 66 to 75% of the total ATP is formed nonoxidatively. The remaining 25 to 35% of the ATP formation is related to the oxidation of NADH. The formation of ATP in the oxidative reaction can be prevented by excluding oxygen or adding cyanide to prevent NADH oxidation. It can also be prevented by adding methylene blue or pyruvate, which bypasses electron transport to oxygen, but does not interfere with NADH oxidation. Potential sources of ATP, such as glycolysis, the pyruvate oxidase reaction, or the oxidative pentose cycle, are not present, and the high nonoxidative ATP formation is ascribed to the adenylate kinase reaction. The reaction requires adenosine diphosphate (ADP) as a phosphate acceptor. NADH oxidation is independent of ADP. Antimycin A, amytal, and 2,4-dinitrophenol decreased, but did not prevent, oxidative formation of ATP. P:O ratios ranged from 0.15 to 0.25. All of the oxidative activity was in the soluble portion of the cell-free extracts.     

Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase of Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.     

Adenosine Phosphate and Nicotinamide Adenine Dinucleotide Pool Sizes during Shoot Initiation in Tobacco Callus

Shoot-forming tobacco callus is found to have high levels of adenosine phosphates and NAD⁺, and a low energy charge during meristemoid and shoot primordium formation. NADH levels are low and show little change during this period. There is a decline in the content of NADPH to nondetectable levels during the process, and a transient increase of NADP⁺ is observed early in culture. These patterns are indicative of a shift to a more intensive rate of metabolism during meristemoid and shoot primordium formation and apparently reflect the requirement for energy and reducing power during organ initiation.     

Synthesis of Methylene-Bridged Analogues of Nicotinamide Riboside,Nicotinamide Mononucleotide and Nicotinamide Adenine Dinucleotide

Abstract

5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose (11a) and ?3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (11b) were prepared by condensation of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-3-pentulofuranose (10) with lithiated (LDA) 2-methylnicotinamide and 6-methylnicotinamide, respectively, and then deprotected to give 1,2-O-isopropylidene-3-(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose(12a) and 1,2-O-isopropylidene-3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (12b). Benzoylation as well as phosphorylation of compounds 12 afforded the corresponding 5-O-benzoate (13b) and 5-O-monophosphates (14a and 14b). Treatment of 13b with CF₃COOH/H₂O caused 1,2-de-O-isopropylidenation with simultaneous cyclization to the corresponding methylene-bridged cyclic nucleoside - 3′,6-methylene-1-(5-O-benzoyl-β-D-ribofuranose)-3-carboxamidopyridinium trifluoro-acetate (8b) - restricted to the “anti” conformation. In a similar manner compounds 14a and 14b were converted into conformationally restricted 2,3′-methylene-1-(β-D-ribofuranose)-3-carboxamidopyridinium-5′-monophosphate (9a - “syn”) and 3′,6-methylene-1-(β-D-ribofuranose)-3-carboxamido -pyridinium-5′monophosphate (9b - “anti”) respectively. Coupling of derivatives 12a and 12b with the adenosine 5′-methylenediphosphonate (16) afforded the corresponding dinucleotides 17. Upon acidic 1,2-de-O-isopropylidenation of 17b, the conformationally restricted P¹-[6,3′-methylene-1-(β-D-ribofuranos-5-yl)-3-carboxamidopyridinium]-P²-(adenosin-5′-yl)methylenediphosphonate 18b -“anti” was formed. Compound 18b was found to be unstable. Upon addition of water 18b was converted into the anomeric mixture of acyclic dinucleotides, i. e. P¹-[3(R)-nicotinamid-6-ylmethyl-D-ribofuranos-5-yl]-P²-(adenosin-5′-yl)-methylenediphosphonate (19b). In a similar manner, treatment of 17a with CF₃COOH/H₂O and HPLC purification afforded the corresponding dinucleotide 19a.

     

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⁺.     

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.     

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.     

烟酰胺腺嘌呤二核苷酸激酶研究进展

本文介绍了ＮＡＤ￣＋激酶的来源和分布、测定方法、最适ｐＨ、反应平衡常数、影响酶稳定性的因子、比活力和动力学常数、三磷酸核苷酸特异性、对金属离子的需要、抑制剂和底物类似物、反应机制、反应的感光性、酶固定化、与钙调蛋白的关系、各种效应物等方面研究的进展,并进行了评述。     

Further Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate:Nitrate Oxidoreductase in Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.     

Correlation Between Reduced Nicotinamide Adenine Dinucleotide Phosphate Levels and Morphological Changes in Neurospora crassa

A colonial mutant of Neurospora crassa, previously shown to be altered in the structure of glucose-6-P dehydrogenase [a reduced nicotinamide adenine dinucleotide phosphate (NADPH) producing reaction], contained only 40% as much NADPH in extracts as did the wild type. A partial revertant strain, when grown at 23 C, had the same total NADPH content as the wild type, but, at 34 C, had lower levels of NADPH as well as a colonial morphology. A revertant with complete wild-type morphology had wild-type levels of NADPH. Two different colonial mutants, which have also been reported to be altered in NADPH-generating reactions, were found to have a lower content of NADPH, whereas other colonial mutants had wild-type levels. The wild-type strain, when grown under conditions in which it contained a lower total content of NADPH, had a morphology similar to that of a colonial mutant. The evidence indicates that lowered NADPH content leads to a dramatic alteration in the morphology of Neurospora, but not necessarily vice versa. The possible pleiotropic effects of the NADPH deficiency are discussed.     

Amperometric Detection of Reduced Nicotinamide Adenine Dinucleotide Using a Film-covered Organic Salt Electrode

A new N-acylated serinol, inconspicamide (1), was isolated from the marine sponge, Stelletta inconspicua, together with a glyceryl ether (2). Their structures were determined on the basis of spectroscopic data and the modified Mosher analysis. They exhibited moderate cytotoxic activity against HeLa human cervical cancer cells.     

Influence of Gas Environment on Catabolic Activities and on Reoxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate in Chlamydia

We investigated the effect of the gas environment on the enzymatic reactions of intact isolated cells of the agents of trachoma and of meningopneumonitis of the host-dependent genus Chlamydia. In comparison with the reactions taking place in a gas phase of air, O(2) depressed CO(2) production from pyruvate and glutamate by trachoma and from glutamate by meningopneumonitis. O(2) enhanced the degradation of pyruvate by meningopneumonitis, but this effect was due to increased H(2)O(2), and was reversed by added catalase. Both dehydrogenation of alpha-ketoglutarate and was reversed by added catalase. Dehydrogenation of alpha-ketoglutarate by both agents and production of CO(2) from C(1) of glucose-6-phosphate were stimulated by O(2) and depressed in N(2). The latter activity was stimulated in air, O(2), and N(2) by nicotinamide adenine dinucleotide phosphate (NADP) in relation to the amount added, and also in air or O(2), but not in N(2), by moderate amounts of NADP and an excess of oxidized glutathione with concomitant formation of H(2)O(2). A small but significant amount of O(2) was consumed during the course of these reactions. It is suggested that glutathione reductase activity can occur only when accompanied by an oxidative reaction, and that this close link between the two reactions represents a mechanism of electron transport which transfers hydrogen to molecular O(2).     

Effects of Hydroxybenzoic Acids on Oxidation of Reduced Nicotinamide Adenine Dinucleotide by Enzymes from Tobacco Leaves

Hydroxyhenzoic acids were tested for their effects on oxidation of the reduced nicotinamide adenine dinucleotide (NADH) in the absence of added H₂O₂ and Mn²* by an enzyme preparation from tobacco leaves (Nicotiana tabacum, var. White Gold). For comparison, a commercial horseradish peroxidase was also used. The rate of NADH oxidation was followed spectruphotometrically at 340 nm. Mono- and dihydroxybenzoic acids exerted significant effect on the rate of NADU oxidation, yet their effectiveness was determined by the number and position of the hydroxyl group on the ring. 4-Hydroxybenzoic acid was very effective in stimulating the reaction. Shifting the hydroxyl from the 4- to the 3-position and from the 3- to the 2-position decreased activity. 2,4- And 2,5-dihydroxybenzoic aeids were more active than the other dihydroxy-iscuners in stinulating oxidation of NADH. the dihydroxybenzoic acids with the hydroxyls in adjacent positions were less effective, and their activity was affected by other phenolic activators. In the presence of 4-hydroxybenzoic acid which enhanced oxidation of NADH, 2,4- and 2,5-dihydroxybenzoic acids further stimulated the reaction, but 3,4-, 2,3- and 2,6-dibydoxybenzoic acids were inhibitory. The inhibition by 3,4- and 2,3-dihydroxybenzoic aciils was non-competitive. The enzymes extracted by a L-cysteine-containing buffer showed lower NADH-oxidase activity. The enzyme preparation possessed peroxidase activity. The activity of NADH-oxidase inereased when H₂O₂ and Mi²* were present in addition to 4-hydroxy-benzoic acid. The effect of the position and number of hydroxyl substitution on the rate of NADH oxidation by borseradish peroxidase was also significant. This suggests the involvement of peroxidase in the NADH-oxidase system of tobacco leaves. However, a combination of the inactivated enzyme solution and active horseradish peroxidase with peroxidase activity equivalent to that of the enzyme preparation from tobacco leaves did not reconstitute the NADH-oxidase activity of tobacco leaves. This and other evidence suggests that the soluble NADH-oxidizing zyme system of tobacco leaves is more complicated than peroxidase.     

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.     

Electron Transport in Halophilic Bacteria: Involvement of a Menaquinone in the Reduced Nicotinamide Adenine Dinucleotide Oxidative Pathway

The reduced nicotinamide adenine dinucleotide oxidative pathway of a halophilic bacterium was found to contain a light-sensitive (360 nm) compound, menaquinone-8, which serves as a cofactor in the nicotinamide adenine dinucleotide⁺-linked pathway.     

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.     

Divalent Cation Activation of Deoxycholate-Solubilized and -Inactivated Membrane Reduced Nicotinamide Adenine Dinucleotide Oxidase of Bacillus megaterium KM

After treating Bacillus megaterium KM membranes with 0.2% sodium deoxycholate, most of the membrane reduced nicotinamide adenine dinucleotide (NADH) oxidase was inactivated, and all of the membrane NADH-2,6 dichlorophenol indophenol oxidoreductase was solubilized. Dilution of the deoxycholate-treated membranes in the presence of divalent cations restored almost all of the original membrane NADH oxidase. The effectiveness of the divalent cation activation decreased in the order Ba(2+) > Ca(2+) > Mg(2+) > Mn(2+). After centrifugation, the deoxycholate-treated membranes at 100,000 x g for 1 hr, all of the NADH oxidase that was activated by a divalent cation was soluble. Cation-activated oxidase, however, was insoluble. The results show that 0.2% deoxycholate at least partially solubilizes the total electron chain from NADH to O(2) in an inactive from which can be reactivated by divalent cations with the formation of active, insoluble NADH oxidase.     

Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva

An NADP⁺-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella.     

Reduction of Activity of Reduced Nicotinamide Adenine Dinucleotide Oxidase by Divalent Cations in Cell-Free Extracts of Bacillus cereus T

A rapid and effective method was devised for the reduction of activity of reduced nicotinamide adenine dinucleotide (NADH) oxidase in crude extracts of Bacillus cereus T. The addition of 25 mumoles of MnCl(2) per mg of extract protein in tris(hydroxymethyl)aminomethane-hydrochloride buffer reduced NADH oxidase activity by 90% within 1 min, and this reduction was independent of pH between pH 7.0 and 8.5. Other divalent cations such as Mg(2+), Ba(2+), Ca(2+), and Co(2+) also reduced NADH oxidase activity, but monovalent cations such as Na(+) and K(+) were ineffective. The reduction of NADH oxidase activity by divalent cations was presumably due to the removal of an essential flavine cofactor, since the addition of riboflavine and flavine mononucleotide to treated extracts was shown to completely restore NADH oxidase activity. The specificity, convenience, and efficiency of the procedure were shown to be applicable to crude extracts of B. megaterium and B. subtilis and should facilitate spectrophotometric measurements of nicotinamide adenine dinucleotide-dependent dehydrogenases in these and other microorganisms.