Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

NAD⁺ levels in resting human lymphocytes obtained from 20 donors were found to be 69.9 ± 21.7 pmols/10⁶ cells. After 3 days of phytohemagglutinin (PHA) stimulation the NAD⁺ levels rose to 452 ± 198 pmols/10⁶ cells. NADH, NADP⁺ and NADPH also increased in mitogen-stimulated lymphocytes, but the major portion of the increase in total pyridine nucleotide pools was accounted for by the increase in NAD⁺. When PHA-stimulated lymphocytes were incubated in nicotinamide-deficient growth medium, there was no significant increase in their total pyridine nucleotide pools; however, the ratios of oxidized to reduced pyridine nucleotides changed in a similar fashion to cells grown in medium containing nicotinamide. When lymphocytes in nicotinamide-deficient medium were stimulated with PHA they increased their levels of DNA synthesis and cell replication in a similar fashion to cells growing in nicotinamide-supplemented media. Human lymphocytes were able to synthesize pyridine nucleotides from nicotinamide or nicotinic acid; however, in the absence of a preformed pyridine ring they did not efficiently use tryptophan for the synthesis of NAD. Uptake of [carbonyl-¹⁴C]nicotinamide and conversion to NAD was markedly increased in PHA-stimulated lymphocytes; these cells also showed a marked increase in activity of the enzyme adenosine-triphosphate-nicotinamide mononucleotide (ATP-NMN) adenylyl transferase.     

Characteristics of 8-substituted adenine nucleotide derivatives utilized in affinity chromatography.

Improved methods for the preparation of several 8-substituted adenine nucleotide derivatives are described. Enzymatic properties of these 8-substituted derivatives were investigated by steady state kinetic and inhibition studies. It was found that 8-(6-aminohexyl)-amino DPN⁺ and TPN⁺ exhibit relatively high affinity for most DPN⁺ and TPN⁺ dependent dehydrogenases. Preliminary nmr studies indicate that the 8-substituted adenine nucleotide derivatives may exist in slightly different ribosyl as well as glycosyl conformations from those of the natural adenine nucleotides. The chemical shift difference between geminal C₄ protons of dihydropyridine moiety of DPN⁺ and TPN⁺ changes from 0.1 to 0.2 ppm upon the 8-hexyl substitution of the natural coenzymes, indicating a strong interaction between 8-hexyl side chain of adenine moiety and the dihydropyridine moiety of these coenzyme derivatives. However, the folding and fluorescence properties of 8-(6-aminohexyl)-amino DPN⁺ and TPN⁺ as well as their reduced analogs in aqueous solutions are not significantly altered as compared to those of natural DPN⁺ and TPN⁺. Purification of glucose-6-phosphate dehydrogenase from yeast extracts and human erythrocytes using 8-(6-aminohexyl)-amino-TPN⁺ -Sepharose column is reported. Preliminary studies on the purification of various kinases using 8-substituted ADP and ATP Sepharose columns are also presented.     

Influence of hormones on the nicotinamide nucleotide coenzymes of adipose tissue

The concentrations of the oxidized and reduced forms of the nicotinamide nucleotides were measured in the epididymal fat pads of normal, alloxan-diabetic and hypophysectomized rats. In both alloxan-diabetic rats and hypophysectomized rats the weight of the adipose tissue fell, as did the total content of NADH and NADPH; in addition, NAD⁺ was decreased in the alloxan-diabetic group. Of these changes the most marked was in NADPH and this was the only significant difference when the results were expressed as nicotinamide nucleotides/mg. of tissue protein. The concentration of NADPH in the hypophysectomized rats was not altered by treatment with growth hormone but was restored to normal by treatment with thyroxine. These results are discussed in relation to the known effect of these hormonal conditions on lipid synthesis in adipose tissue.     

The pyridine nucleosidase from Bacillus subtilis. Kinetic properties and enzyme-inhibitor interactions.

The interaction between the Bacillus subtilis DPNase and its inhibitor is a time-dependent second order reaction, with a rate constant of 5.3 × 10⁵ M^?1 s^?1 at 28 °C and pH 7.5. The interaction is noncompetitive with the substrate, and the presence of substrate does not affect the rate of interaction. Dissociation of the enzyme-inhibitor complex occurs below pH 4.3. The presence of DPN⁺ does not promote dissociation of the complex at neutral pH values, but does promote dissociation at pH 4.5.The enzyme has a high specificity for the presence of the nicotinamide ring in the substrate. DPN⁺ and TPN⁺ are the only dinucleotides that are hydrolyzed by the DPNase out of a number of DPN⁺ analogs that were tested. The thionicotinamide analog of DPN⁺ is a potent inhibitor of the enzyme.The stability of the DPNase and its inhibitor against heat and acid denaturation was also investigated.     

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in yoshida ascites hepatoma AH 130

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD⁺) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP⁺ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP⁺ plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucroselow salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucrose-low salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Hexose transport derepressed and refractory to purine regulation in NAD(H)-depleted Nil cells

Nil hamster fibroblasts depleted of NAD(H) by growth in medium devoid of nicotinamide (NAm^?MEM) exhibit up to 2-3-fold higher rates of glucose transport. Derepression of glucose transport is observed only when Nil cells have become severely depleted of both intracellular NAD(H) and ATP, despite the continued presence of 5.5 mM D-glucose in the growth medium. Neither the initial rate of transport, approximated from 3-O-methylglucose uptake, nor accumulation of D-glucose itself is repressed upon restoring nicotinamide to the medium. Exposure of the cells to NAD⁺ (10^?5 M), however, leads to a sharp curtailment of transport within 2 to 3 hours. The purines, hypoxanthine and guanine, that sharply reduce glucose transport capacity of normal cells, have no significant effect upon transport activity of NAD(H)-depleted cells.     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

Analysis of the effects of ethanol, fructose and nicotinamide on the free nucleotides of rat liver using high performance liquid chromatography

1. Rat liver nucleotides were separated on a weak anion-exchange HPLC column. The characteristic nucleotide profile could be modified by treatment of the rats with ethanol, at both acute and chronic dosages, by fructose and by nicotinamide. 2. The major effect observed after ethanol administration was a decrease in the concentration of ATP with increases in the concentrations of AMP and other nucleotide monophosphates. 3. These changes gradually reverted to normal values over a 30 min period. 4. Ingestion of 1 or 5% ethanol for 4 weeks caused similar changes in the nucleotide profile of liver. 5. Fructose administration caused a dramatic but reversible decrease in the size of the entire nucleotide pool. 6. Rats given daily injections of nicotinamide exhibited greatly elevated concentrations of liver NAD+, whereas the other pyridine nucleotides were relatively unaffected. 7. The results are discussed in relation to the known effect on metabolism of the compounds tested.     

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.     

Preliminary evidence for a pyridine nucleotide cycle in Bordetella pertussis

Preliminary evidence that Bordetella pertussis has a functional pyridine nucleotide cycle was the observation that [¹⁴C]-nicotinic acid was rapidly metabolized during its uptake by the bacteria to pyridine nucleotides and nicotinamide. Nicotinamide deamidase activity, necessary for the completion of the cycle by conversion of nicotinamide to nicotinic acid, was found in a soluble extract (20000 x g supernatant) of B. pertussis cell lysates.This work was supported by the Science Research Council and Wellcome Research Laboratories.     

The role of the pentose phosphate shunt in glucose-induced insulin release: In vitro studies with 6-aminonicotinamide, methylene blue, NAD, NADH, NADP, NADPH and nicotinamide on isolated pancreatic rat islets

The possible role of the pentose phosphate shunt in insulin release was investigated in vitro with collagenase isolated pancreatic islets of rats. Parameters measured were insulin released into the medium and measured by an immunoassay and formation of ¹⁴CO₂ from glucose labeled either in the C-1 or C-6 position. The in vitro effect of the following substances was studied:

1. 1. 6-Aminonicotinamide, an antimetabolite in the synthesis of pyridine nucleotides. In islets of animals pretreated with 6-amino nicotinamide 6 h previously and in the presence of 3 mg/ml glucose in the incubation medium, 6-aminonicotinamide markedly reduced oxidation of [1-¹⁴C]glucose but did not affect that of glucose labeled in C-6. Concomitantly there was a marked decrease in insulin release. This action of 6-aminonicotinamide did not take place when it was added only to the incubation medium. Pretreatment with 6-aminonicotinamide did not change the insulin concentration of the islets, making it unlikely that it interfered with insulin synthesis. The effect of 6-aminonicotinamide is consistent with partial inhibition of the pentose shunt.

2. 2. Methylene blue: this agent was selected because it is known from studies with red blood cells that it will oxidize NADPH and thus stimulate activity of the pentose shunt. In concentrations of 0.5 and 2 μg/ml, methylene blue markedly stimulated oxidation of [1-¹⁴C]glucose but not that of C-6. Simultaneously there was a dose related decrease of insulin released.

3. 3. Pyridine nucleotides: in the absence of glucose only NADPH exhibited a significant effect of insulin release. If glucose (3 mg/ml) was present 1 or 10 mM of NAD⁺ or NADH exhibited a significant effect, NADP⁺ or NADPH were less effective. If the pentose shunt was blocked by pretreatment with 6-aminonicotinamide, all 4 pyridine nucleotides stimulated insulin release. Similarly there was an increase in oxidation of [1-¹⁴C]glucose, consitent with restimulation of the pentose shunt.

4. 4. Nicotinamide by itself exhibited a small effect; however, it was much less than the one produced by equimolar concentrations of the pyridine nucleotides.

Conclusion: Restricted availability of NADPH either less production or by fast removal leads to a decrease in glucose-induced insulin release. Pyridine nucleotides will restimulate 6-aminonicotinamide blockade insulin release and glucose oxidation by the pentose shunt. Recently it has been proposed by others that the polyol pathway may play a key role in insulin release, our data are consistent with such a hypothesis. Furthermore they do support a major role of the pentose shunt in insulin release.     

Deprival of nicotinamide leads to enhanced glucose transport in chick embryo fibroblasts

Chick embryo fibroblasts growing in medium free of pyridine ring precursors of NADH and NADPH replicate several times before multiplication ceases. The rate of glucose transport is progressively enhanced, finally reaching levels several times higher than those normally observed in cells severely depleted of NADH. Whereas normal cells respond to additional glucose by further reducing transport, the NADH-depleted cell is refractory to glucose even at five times the normal glucose concentration. Readdition of nicotinamide does little to restore normal transport within 24 h. On the other hand NAD+ or NADP+ provided simultaneously with glucose results in a sharp decline in measurable transport within 2-4 h. The role of the pyridine nucleotides in this reduction of transport function is for the moment unknown.     

Nicotinamide nucleotides in the erythrocytes of patients suffering from pellagra

1. The nicotinamide nucleotide concentrations in the erythrocytes of subjects suffering from pellagra (pellagrins) were not lower than those in normal subjects, but the ability of erythrocytes to synthesize these nucleotides in vitro was significantly lower in pellagrins. 2. The oral administration of 10g. of l-leucine daily for 5 days depressed the nicotinamide nucleotide-synthesizing ability of erythrocytes both in normal subjects and in pellagrins. This was not accompanied by changes in the nucleotide concentration in erythrocytes. 3. Quinolinic acid brought about a significant inhibition of the synthesis of nicotinamide nucleotides in vitro. Such inhibition was partially overcome by increasing the concentration of nicotinic acid in the medium.     

Identity of V factor in culture medium used for prior growth of two strains of Staphylococcus aureus.

Staphylococcus aureus ATCC 12598 and ATCC 25923 were starved of pyridine nucleotides and precursors and then grown in a semidefined medium containing [carbonyl-14C]nicotinamide. Samples of medium from late-exponential-phase and stationary-phase cultures were analyzed for 14C-metabolites. In all cases, V factor was present primarily as NAD.     

Autoradiographic studies of pyridine nucleotide metabolism in human culture cells

More than 80% of the intracellular pyridine metabolite pool of human culture cells is trapped by OsO₄ fixation. The fixed pyridine metabolites fully exchange with nicotinamide and nicotinic acid but not with nicotinamide adenine dinucleotide. Yet, chromatography of the exchanged compounds reveals that NAD and NADP constitute more than 95%. of the fixed material. Although the mechanism of OsO₄ fixation is not fully understood, such fixation has permitted the autoradiographic detection of intracellular pyridine metabolites. Cells of the human cell line, D98/AH2, synthesize pyridine nucleotides during all phases of the cell cycle at rates which do not vary by more than six-fold. There is no difference in the apparent concentration of pyridine metabolites between nucleus and cytoplasm after ten minute or three day pulses with ³H-nicotinic acid. The ³H-labeled pyridine ring is lost from D98/AH2 cells upon transfer to unlabeled medium. In general, the rate of loss is uniform among cells in the population. However, in a small proportion of cells there is little or no loss. Non-dividing cells lose the pyridine ring at approximately the same rate as dividing cells, yet the intracellular concentration of pyridine metabolites is 50% greater in non-dividing cells.     

Effects of growth conditions on the ion composition of Bifidobacterium bifidum subsp. pennsylvanicum

The cation content of Bifidobacterium bifidum subsp. pennsylvanicum was markedly influenced by the washing procedure of the cells, by the growth phase and the temperature, and by the composition of the culture medium. Optimal retention of cations was achieved by washing with 0.25 M MgCl₂ at 20 C. The intracellular Na⁺ concentration rose during growth in normal medium to a constant value in the stationary phase, the K⁺ concentration rose in the exponential phase, but fell in the stationary phase. Cells from 29-C cultures contained more Na⁺ and less K⁺ in the stationary phase than did cells from 37-C cultures, but the total cation content was the same at 29 and 37 C.Intracellular Na⁺ and K⁺ concentrations were dependent on the concentrations in the medium and on its osmolarity. The intracellular Na⁺/K⁺ ratio varied from 0.04 to 2.3. The concentrations of Na⁺, K⁺ and phosphate in the medium hardly affected growth. Mg²⁺-deficiency of the medium markedly decreased the concentration of Mg²⁺ within the cell; its concentration in the cell sap was greatly affected, but the amount of sedimentable, bound Mg²⁺ only slightly. The content of K⁺ within the cell decreased in Mg²⁺-deficient medium, but the concentration of Na⁺ did not. Omission of Tween 80 as well as its substitution by Tween 20 caused a decrease of intracellular K⁺. Cells from Tween 40 and Tween 60 cultures additionally contained markedly less Na⁺.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

Thyroid hormone action on mitochondria

Measurements of fluorescence at >420 nm and extracted NADPH in mitochondria obtained from the livers of hypothyroid rats show that the addition of Pi, ADP and glutamate rapidly reduces over 90% of the total reducible intrinsic pyridine nucleotides in State 3, compared with 20% in normals. The total fluorescence intensity change and reducible NADP⁺ is about twice normal in hypothyroid mitochondria. Adding 6–30 µMl-thyroxine to hypothyroid mitochondriain vitro decreases and delays the substrate-induced reduction of pyridine nucleotides, and excludes both NADP⁺ from such reduction and NADPH from oxidation by added ADP + Pi, without changing the high NADP(H) content. The correcting actions of the hormone are rapidly reversed by albumin, probably by binding free hormone. Changes in respiration do not appear to account for these observations. There is indirect evidence for decreased phosphorylation of added ADP in hypothyroid mitochondria, and a correction by added hormone. The hormonal actions on NADP(H) redox reactions are not reproduced by 1 to 6 µM dinitrophenolin vitro.l-Thyroxine appears to specifically block the participation of NADP (H) in redox reactions in mitochondria from hypothyroid rats, perhaps by effecting a sequestration of the nucleotide, by inhibiting the pyridine nucleotide transhydrogenase, or by activating an energy-linked process that competes with transhydrogenation.Papers I–III in this series were published inArch. Biochem. Biophys.I–124 (1968) 238.II–124 (1968) 248.III–150 (1972) 618.This work was supported by grants from the NIH (AM13564) and from The John A. Hartford Foundation.