Nicotinamide Adenine Nucleotide Research Articles

Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances.

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO(2)) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD(+)). Oxidative stress, as a resultof which the NAP(P)(+)/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO(2) to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO(2) levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.

Intracellular flux of glucose metabolism in streptococcal cells by simultaneous monitoring of fluorescence dependent on reduced nicotinamide adenine nucleotide and acid excretion under strictly anaerobic conditions.

Reduced nicotinamide adenine nucleotide (NADH)-dependent fluorescence and acid excretion during glucose pulse to washed Streptococcus mutans cells were monitored simultaneously at pH 7.0 with a fluorescence spectrophotometer and a pH-stat. Acid excretion started with addition of glucose. At the same time, the fluorescence dropped quickly to a minimum level and increased to a plateau level, suggesting that pyruvate metabolism started immediately after addition of glucose, then the rate of the pyruvate metabolism became almost equal to the rate of glycolysis. When the acid excretion stopped, the fluorescence increased rapidly from the plateau to the maximum level, suggesting that the pyruvate metabolism stopped first, and then began to decrease to the original level. The system used in this study for simultaneously monitoring the level of NADH and acid excretion gives us a crucial tool to clarify a biochemical mechanism of the control of sugar metabolism by streptococci.

Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death.

Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of approximately 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed.

Isoform-specific regulation of nitric oxide synthase mRNA in the kidney by sodium and blood pressure.

The roles of nitric oxide synthases (NOS) in kidney function are still controversial, principally due to the lack of isoform-specific inhibitors of NOS. To investigate the relative roles of each isoform of NOS in regulation of sodium and volume homeostasis. We studied the effects of long-term modifications of sodium diet and blood pressure on expression of NOS mRNA in the renal cortex, where the three isoforms of NOS are present. We used quantitative reverse-transcription-polymerase chain reaction assays specific to each isoform of NOS to determine amounts of their respective mRNA in control rats, deoxycorticosterone acetate (DOCA)-salt hypertensive rats, rats fed a high-salt diet, and furosemide-treated rats fed a low-sodium diet. Nicotinamide adenine nucleotide phosphate H (NADPH) diaphorase staining was performed on DOCA-salt and control rat kidneys. Levels of NOS I mRNA in DOCA-salt rats were decreased by treatment, those in low-salt-diet rats remained unaffected and those in high-salt diet rats tended to be intermediate between those of the other rat groups. Expression of NOS III mRNA was not significantly modified by either treatment Levels of NOS II mRNA in DOCA-salt rats were increased, those in high-salt-diet rats remained unaffected, and those in low-salt-diet were decreased by treatment, but these levels are more than 100-fold lower than those observed for the other isoforms of NOS. NADPH diaphorase staining in macula densa of DOCA-salt rats was markedly decreased compared with that in macula densa of control rats but staining in renal inflammatory and fibrous lesions became detectable, and staining in the vessels did not differ from that for control rats. Our results show that intake of sodium and extracellular fluid volume regulate levels of mRNA of the three NOS isoforms in the renal cortex differently, suggesting that each of them plays a specific role.

Treatment of high-risk, refractory acquired methemoglobinemia with automated red blood cell exchange

Ingestion of strong oxidant substances may result in acquired methemoglobinemia, a clinical condition in which the oxidized blood hemoglobin is incapable of delivering oxygen to the tissues, and the patient becomes cyanotic. Traditional first-line therapy consists of infusion of methylene blue, whose action depends on the availability of reduced nicotinamide adenine nucleotide phosphate (NADPH) within the red blood cell (RBC). Some patients, particularly those who are deficient in glucose-6-phosphate dehydrogenase (G6PD), will not benefit from methylene blue. In these patients, and in some patients who have ingested very strong oxidants, methylene blue may also precipitate Heinz body hemolytic anemia. We present a case of severe, acquired methemoglobinemia in a 26-month-old, 9.8-kg boy with G6PD deficiency. He was cyanotic, in respiratory failure, intubated in a pediatric intensive care unit. In typical fashion, he did not respond to methylene blue. Manual exchange of two whole blood volumes, performed over 4 1/2 hr, also failed to resolve his severe methemoglobinemia. An automated RBC exchange (1.3 RBC volume), lowered his methemoglobin content from 31.8% to 7% in a single 40-min procedure. Thereafter his methemoglobin level continued to decrease rapidly and spontaneously. He was discharged home 2 days later, with 0.4% methemoglobin. To our knowledge, this is the first report to demonstrate the (potentially superior) effectiveness of automated RBC exchange for treatment of patients with high-risk acquired methemoglobinemia, that is, those with G6PD deficiency or who have ingested strong oxidants.

Mechanisms of force inhibition by halothane and isoflurane in intact rat cardiac muscle.

1. We investigated the mechanisms underlying the negative inotropic effect of the volatile anaesthetics halothane and isoflurane using twenty-two intact, right ventricular trabeculae of rat. [Ca2+]1 was measured qualitatively using either fluo-3 or fura-2, loaded into the cytosol via the acetoxymethyl (AM) ester form. Diastolic sarcomere length was adjusted to 2.1-2.2 micrograms and experiments were performed at 21-23 degrees C. 2. Halothane (0.25-3%) and isoflurane (0.48-4%) produced dose-dependent decreases in the amplitudes of the intracellular Ca2+ transients and twitch force. When the fluorescent Ca2+ indicator signals were corrected for changes in autofluorescence, neither volatile anaesthetic significantly changed diastolic [Ca2+]. 3. The ability of halothane and isoflurane to induce Ca2+ release from the sarcoplasmic reticulum of quiescent trabeculae was examined. When the superfusate was Ca2+ ad Na+ free (thereby preventing Na(+)-Ca2+ exchange and Ca2+ influx), 2% halothane, but not 4% isoflurane, evoked a transient increase in [Ca2+]i. 4. Halothane and isoflurane produced reversible, dose-dependent changes in cellular autofluorescence, the pattern of which was consistent with an increase in concentration of the reduced forms of nicotinamide adenine nucleotides and flavoproteins. This observation supports the putative inhibitory action of volatile anaesthetics at the site of Complex I of the mitochondrial electron transport chain. 5. Addition of the fatty acid hexanoate, a substrate that can be metabolized in the face of Complex I inhibition, did not appreciably attenuate the anaesthetic-induced negative inotropy; however, it greatly diminished autofluorescence changes. 6. To determine whether direct actions of the volatile anaesthetics on the contractile system contributed to the negative inotropy, external [Ca2+] was varied to modulate the amplitude of the Ca2+ transient. In the presence of 2% halothane or 4% isoflurane, restoration of the peak Ca2+ transient to control levels did not restore peak force. Moreover, halothane (1%) and isoflurane (16%) each reduced maximal Ca2(+)-activated force (attained using ryanodine tetani and a high external [Ca2+]) by around 15%. 7. We conclude that the negative inotropic actions of halothane and isoflurane on intact cardiac muscle reflect both reduced availability of Ca2+ and decreased responsiveness of the contractile system to Ca2+. The inhibitory action of the volatile anaesthetics on mitochondrial function does not contribute significantly to the negative inotropy but may lead to changes in cellular autofluorescence and misinterpretation of fluorescent Ca2+ indicator signals.

Fumarate-enriched blood cardioplegia results in complete functional recovery of immature myocardium

Amino acid enrichment of cardioplegic solutions has been shown to improve both the metabolic and functional recovery of ischemic myocardium. However, because of the marked systemic vasodilatation involved, use of amino acid enrichment is limited to the periods of induction and reperfusion. Fumarate is a Krebs' cycle intermediate whose conversion to succinate is responsible for the generation of adenosone triphosphate and the oxidation of the reduced form of nicotinamide-adenine nucleotide which is the pathway by which aspartate exerts its effect. Fumarate may also function as a free-radical scavenger and is involved in calcium transport. To determine if fumarate-enriched blood cardioplegia would improve the functional recovery of the neonatal heart, 14 neonatal piglet hearts were isolated and placed on a blood-perfused working heart circuit. After the baseline functional and metabolic assessment was done, cold ischemic arrest was initiated with either standard blood cardioplegic solution (group I; N = 7) or fumarate-enriched (13 mmol/L) blood cardioplegic solution (group II; N = 7). Cardioplegic solution was given at a pressure of 40 mm Hg every 20 minutes for 2 hours, and topical hypothermia was used. Sixty minutes after warm whole blood reperfusion, the functional recovery at left atrial pressures of 3, 6, 9, and 12 mm Hg was 70%, 66%, 66%, and 65%, respectively, in group I, versus 102%, 106%, 105%, and 109%, respectively, in group II (p < 0.05). The tissue creatinine phosphate levels after reperfusion were significantly higher in group II hearts (15.0 ± 1.2 μmol/g dry heart tissue) than in group I hearts (9.2 ± 1.9 μmol/g dry heart tissue), although the adenosine triphosphate levels were not significantly different. The addition of fumarate to standard blood cardioplegic solution results in significantly improved (100%) functional recovery compared with that observed for standard blood cardioplegic solution.

EVALUATION OF SOLUTIONS FOR SMALL INTESTINAL PRESERVATION

A number of organ preservation solutions have been formulated to slow the inevitable progression of ischemic injury, thus prolonging the storage time between removal and implantation. As adenine nucleotide content has been shown to correlate with the functional recovery of transplanted livers and hearts, this study investigated the effects of 24 hr of storage in three preservation solutions, saline (SA), Euro-Collins (CO), and University of Wisconsin (UW) on adenine and nicotinamide adenine nucleotides and inosine content of the rat small intestine. Significant biochemical differences were found between segments as early as after the initial perfusion when the inosine content was higher in UW-perfused than CO- or SA-perfused segments. After 2 hr of storage in CO solution and after 6 and 24 hr in both CO and UW solutions, the ATP content was higher than in SA-stored segments. In addition to inosine, which was significantly higher at all time points for UW-stored segments, the AMP and total adenine nucleotide content of UW-stored segments at 24 hr was significantly higher than SA- or CO-stored segments. After 24 hr of storage, those segments stored in UW were able to utilize significantly more oxygen than SA-stored. These data provide biochemical evidence supporting the advantages of CO and UW storage solutions over SA for preservation of small intestine segments.

A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis

Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a verscolorin A-accumulating mutant, NIAH-9, resulted in accumulation of versiconol acetate (VOAc) and versiconal hemiacetal acetate (VHA), whereas the production of aflatoxins, versicolorin A (VA), and versiconol (VOH) decreased. In feeding experiments using another non-aflatoxigenic mutant, NIAH-26, aflatoxins were newly produced from each of VHA, VOAc, VOH, versicolorin B (VB) and versicolorin C (VC). In these experiments, aflatoxin production from VHA or VOAc was inhibited by dichlorvos, whereas that from each of VOH, VB and VC was insensitive to dichlorvos. In cell-free experiments using the cytosol fraction of NIAH-26, VHA was converted to VC (or VB) and a substance tentatively identified as versiconal (VHOH). By further addition of NADH or NADPH to the same reaction mixture, VOAc and VOH were also formed together with VC (VB) and VHOH. VOH was produced from VOAc irrespective of nicotinamide adenine nucleotide. Also, the incubation of VOH in the presence of NAD or NADP led to the formation of VC (VB). The production of VC (VB) and VHOH from VHA, and that of VOH from VOAc was inhibited by dichlorvos, whereas the production of VOAc from VHA, and that of VC (VB) from VOH, was insensitive to dichlorvos. These results indicate that a metabolic grid catalysed by dehydrogenase and esterase among VHA, VOAc, VOH and VHOH, and a reaction from VHOH to VC (VB) are involved in aflatoxin biosynthesis. These enzyme activities were also detected when yeast extract peptone medium was used, or when A. oryzae SYS-2 was examined.

Mechanism of fatty acid effect on myocardial oxygen consumption. A phosphorus NMR study

The calorigenic effect of fatty acids on the intact myocardium was investigated in isolated rat hearts perfused with a phosphate-free bicarbonate buffer. 31P-NMR spectra were accumulated in a Fourier transform high-field spectrometer during titration of the heart with varying concentrations of hexanoate. Oxygen consumption, coronary flow, left ventricular pressure development, heart rate and tissue surface fluorescence of nicotinamide-adenine nucleotides were monitored simultaneously. It was found that hexanoate within a range of 40 to 800 μM increased the reduction of NADH/NAD, increased oxygen consumption and increased the phosphocreatine/inorganic phosphate ratio in a concentration-dependent manner. The results suggest that the fatty acid-induced increase in oxygen consumption is not due to a primary effect on the cellular energy state resulting from a decrease in the P O ratio or uncoupling of the mitochondria but due to an increase in the thermodynamic driving force.

Effect of ethanol on cell growth, metabolism and cation fluxes in the yeast Metschnikowia reukaufii

The yeast Metschnikowia reukaufii could not utilize ethanol as a growth substrate, although ethanol stimulated cellular O2 consumption and the reduction of intracellular nicotinamide adenine nucleotides. Also, ethanol inhibited cell growth in glucose-containing medium. The effect was stronger when the K+ concentration in the growth medium was lowered from 5.8 to 0.6 mM. Addition of glucose to an aerated cell suspension caused an initial H+ efflux and K+ influx in a ratio of approximately 1:1. This was followed by a phase of continuing extracellular acidification without any measurable uptake of K+. In contrast, in cells energized by glucose, ethanol stimulated K+ efflux; concomitantly, H+ extrusion was markedly lowered by ethanol. The rates of H+ extrusion correlated with the intracellular level of glucose 6-phosphate and not of ATP. It is concluded that there is a regulatory interaction, though not by a direct effect, between glucose 6-phosphate and the plasma-membrane ATPase. Ethanol appears to activate electron transfer from cytosolic NADH to O2 by a pathway independent of the mitochondrial respiratory chain.

Atrial bioenergetic variations in moderate hypoxia: danger or protective defense?

The effects of hypoxia on contractile tension and on tissue adenylate pool content, nicotinamide adenine nucleotide, NAD, nicotinamide adenine dinucleotide phosphate, NADP, and creatine phosphate, CrP, were investigated in isolated, spontaneously beating, guinea pig atria. When two different degrees of hypoxia were induced by lowering oxygen tension from 95% O2 (control) to 40% (moderate hypoxia) and 20% (severe hypoxia) for 30 min, contractile tension slowly decreased to 60% and 40% of control, respectively. In 40% O2 hypoxic atria, ATP was not significantly decreased, AMP slightly increased, TAN (total adenylate nucleotides) and adenylate energy charge [(ATP + 0.5 ADP)/(ATP + ADP + AMP)] did not change and creatine phosphate was decreased down to 53%. Hypoxic atria in 20% O2 showed a significant decrease of 26% in ATP, while ADP and AMP increased four and seven times, respectively. The adenylate energy-charge value was reduced from 0.93 to 0.70. Creatine phosphate decreased to below the analytical detection limit. Moderate hypoxia (40% O2), which induced a significant decrease of contractile tension but only minor changes of energetic tissue metabolism, was further investigated 2, 5, and 10 min after low oxygen tension was applied. Two stages of variations were evident during 30 min of experimental hypoxia. Within the first 10 min, concomitantly with atrial tension decrease, ATP, NAD, NADP, ATP/AMP, ATP/ADP, and TAN decreased, CrP began to decrease, inosine and xanthine showed no significant change. During the following 20 min of hypoxia, all parameters returned to the control levels with the exception of creatine phosphate. Adenylate energy charge did not change. The electrophysiological analysis of atrial cells did not show any major change in action potential configuration and resting potential, during 40% O2 hypoxia. The differences at metabolic level between moderate and more severe hypoxia suggest that the energetic state may be extremely unbalanced, in atrial tissue, as long as hypoxia is aggravated. Moreover, the time-course study, during 30 min of 40% O2, suggests that the early decrease of contractile tension does not depend on lowered energy availability, instead it might be, at least in part, a preventive measure to maintain energy balance in myocardial tissue to counteract hypoxic damage and, in this mechanism of defense, creatine phosphate shuttle seems to play a relevant role.

Establishment and characterization of long-term cultured cell lines of murine resident macrophages.

Murine resident macrophages can proliferate in vitro when they are grown in coculture on a layer of mesothelial or endothelial type feeder cells. Resident macrophages were obtained from lung explants of C57Bl/6 lpr/lpr mice and from spleen explants or peritoneal washing of Balb/c mice; the cells were seeded without further washing. After 3-4 weeks of culture, the macrophages began to proliferate on a confluent layer of feeder cells. The macrophages then could be collected in the fluid phase and reseeded for permanent culture after generation of a new feeder layer. These cells were characterized as macrophages by the following criteria: 1) their morphology, ultrastructure, and adherence properties; 2) more than 90% of the macrophages phagocytized yeasts compared with less than 1% of the feeder cells; 3) the presence of functional Fc and mannose receptors, nonspecific cytoplasmic esterases, and membrane ectoenzymes such as nicotinamide adenine dinucleotide (NAD) glycohydrolase and nucleotide pyrophosphatase; 4) by cytofluorographic phenotype analysis with monoclonal antibodies, characterizing a normal macrophage population (MAC1+, Fcrec+, H-2K+, THY1-, LYT2-, L3T4-). 5) by functional studies proving that the expanded macrophages could function as accessory cells in the induction of lymphocyte proliferation in response to concanavalin A (Con A), that they generated reactive oxygen radicals and that they were cytotoxic for tumor cells. During coculture, growth or activating factors such as macrophage colony-stimulating factor or gamma-interferon were released in the medium. Long-term cultured macrophages had chromosomal abnormalities. Our study suggests that tissue macrophages can proliferate in vitro and hence that it is possible to establish long-term cultured cell lines of macrophages of defined and reproducible characteristics.

Purification of bovine liver S-adenosylhomocysteine hydrolase by affinity chromatography on blue dextran-agarose

S-Adenosylhomocysteine (AdoHcy) hydrolase (adenosylhomocysteinase, EC 3.3.1.1) was purified from bovine liver by conventional protein purification procedures (differential centrifugation, ammonium sulfate fractionation and DEAE-cellulos chromatography) followed by affinity chromatography on blue dextran coupled to agarose. The enzyme was eluted from the blue dextran-agarose column with adenosine and the adenosine removed by chromatography on Sephasex G-75. The affinity chromatography step resulted in a substantial increase in total AdoHcy hydrolase activity (about 600%) suggsting either removal of some inhibitory substance or a change in the structure of the protein producing a more catalytically efficient enzyme. The isolation procedure afforded over 3400-fold purification of the enzyme, which was shown to be homogeneous by polyacrylamide gel electrophoresis. Using high pressure liquid chromatography, the nucleotide content of the freshly purified enzyme was determined to be 2 mol of nicotinamide adenine nucleotide per mol of enzyme tetramer. The ratio of the reduced to the oxidized form of the nucleotide was correlated to the activity of the enzyme preparation.

Binding of NAD and NADP dimers to NAD- and NADP-dependent dehydrogenases

Interaction of the electrolytically prepared dimers of nicotinamide adenine nucleotide, (NAD) 2, and nicotinamide adenine nucleotide phosphate, (NADP) 2, with lactate, alcohol, glyceraldehyde 3-phosphate, α-glycerophosphate, glutamate and glucose-6-phosphate dehydrogenase has been studied using the quenching of protein fluorescence, kinetics of inhibition and the stopped-flow method. It has been shown that these enzymes are able to bind dimers preserving their coenzyme specificity. The most efficient binding of (NAD) 2 has been observed in the case of glutamate and lactate (bovine heart) dehydrogenase, the dissociation constants being 6 and 8 μM, respectively. (NADP) 2 affinity to glutamate and glucose-6-phosphate dehydrogenase is also fairly high. More detailed studies on the interactions of dimers with alcohol and glutamate dehydrogenase have shown that the binding to the coenzyme binding site is the prerequisite for the association. However, some additional stabilizing interactions with other enzyme groups are not excluded, though (NAD) 2 does not bind to the known binding sites of these enzymes, such as the substrate pocket of alcohol dehydrogenase and the regulatory binding sites for ADP and GTP of glutamate dehydrogenase.

Fluctuation of enzyme activities related to nitrogen fixation, C6/C1 ratio, and nicotinamide nucleotide contents during soybean plant development

Activities of enzymes related to nitrogen assimilation, hexose metabolism, C6/C1 ratios and nicotinamide adenine nucleotide contents were determined in soybean nodules and root tissues during the plant development. Glutamate dehydrogenase and alanine dehydrogenase activities in nodules were more dependent on NADH than NADPH, though the NADPH-dependent activities exhibited higher values in the roots. In both tissues GOGAT activity was NADH-dependent, and G6P dehydrogenase and 6PG dehydrogenase activities were NADP-dependent. These enzyme activities on a mg protein basis showed high levels throughout the plant development and their fluctuations were not correlated with those of nitrogenase activity. Their specific activities, however, exhibited higher values at the early stage of plant development. The C6/C1 ratios in nodule and root tissues were always below 0.6. The value was 0.5 at the early stage, then decreased gradually and increased after the flowering stage. At the early pod filling stage the nodule...

The hydrolysis of nicotinamide adenine nucleotide by brush border membranes of rat intestine.

The hydrolysis of NAD by rat intestine was studied to determine the subcellular site of this hydrolysis and to identify the niacin-containing products that are formed. Using [nicotinamide-14C]NAD as substrate, and high pressure liquid chromatography for identification and quantification of products, the present study demonstrates two independent reactions for the hydrolysis of NAD; one that forms nicotinamide through hydrolysis of the ribosyl-pyridinium bond and one that forms nicotinamide mononucleotide through the hydrolysis of the pyrophosphate bond. The nicotinamide mononucleotide is subsequently dephosphorylated to nicotinamide riboside. Enzymes which release nicotinamide mononucleotide and nicotinamide riboside are associated with the brush border membrane as determined by analysis of fractionated intestinal homogenates. The enzyme activity which releases nicotinamide from NAD is associated with the brush border membrane fraction and also with a second cellular particulate fraction. Between pH5 and pH6 NAD is hydrolysed principally to nicotinamide. At pH 7.0 rates of nicotinamide and nicotinamide mononucleotide formation are the same. Above pH 7.0 the formation of nicotinamide mononucleotide is preferred.

Hybrid organometallic/enzymic catalyst systems: regeneration of NADH using dihydrogen

A mixture of a water-soluble bis(phosphine)rhodium complex and o- and l-lactate dehydrogenase catalyzes the reduction of nicotinamide adenine nucleotide (NAD+) to NADH by Hz in an aqueous solution containing pyruvate (Scheme I). Coupling of this system to the asymmetric reduction of 2-norbornanone using horse liver alcohol dehydrogenase is demonstrated. Organometallic and enzymatic catalysts generally exhibit their most characteristic activities on different classes of reactants: organometallic catalysts are especially effective with nonpolar substances (e.g., Hz, CO, and olefins), while enzymatic catalysts are most effective with polar, polyfunctional materials (e.g., carbohydrates, derivatives of acids, and biopolymers). Combi- nations of organometallic and enzymatic components which in- tegrate these two types of substrate selectivities may have un- common and useful catalytic activities. Here we demonstrate the operation of a hybrid system involving the combination of a water-soluble bis(phosphine)rhodium complex with two enzymes, whose overall action is to catalyze the reduction of NAD+ to NADH by Hr.t The operation of this system and illustrations of its application to enzyme-catalyzed reduction reactions requiring NADH regeneration are summarized in Scheme L The regen- eration of the nicotinamide cofactors (NAD(P)-NAD(P)H) has been the subject of previous research in our laboratories. Several procedures are presently available;ts8 the evaluation of their relative merits has not been completed. Results

Morphology and metabolism of adult rat hepatocytes in primary culture.

Isolated hepatocytes obtained by collagenase perfusion of adult rat livers were seeded on collagen gels and kept in a chemically defined culture medium (except for the first 6 h of culture where 10% fetal calf serum was added). Cells adopted an epitheloid shape within 4 h and arranged themselves in a trabeculae-like pattern during the first 20 h of culture. In the electron microscope numerous tight junctions and bile capillaries were observed at sites of cell-to-cell contact. From metabolite analyses in the culture medium the following conclusions can be drawn: The cells continued to synthesize urea and ketone bodies for 5 days of culture. The cytosolic and mitochondrial redox states of the nicotinamide adenine nucleotide systsm were as in the liver in vivo and the oxygen supply of hepatocytes was sufficient under the culture conditions. Maximal velocities of ketogenesis from octanoate and of urea formation from ornithine plus ammonium chloride were stable during a 120 h culture period and compared well with rates found in the isolated perfused rat liver.

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis

A-Side (4- R)-(4- 2H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol- d 6 to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4- S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1- d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final A 260 A 340 ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2- d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.