Reduced nicotinamide adenine dinucleotide and depolarization in neurons.

AMERICAN JOURNAL OF PHYSIOLOGY Vol. 228, No. 4, April 1975. Printed in U.S.A.

Reduced

nicotinamide

and depolarization

adenine in neurons

CARLOS RODRiGUEZ-ESTRADA Ca’tedra de Fisiologia, Institute de Medicina Experimental, Universidad Central de Venezuela, Caracas, Venezuela

RODRIGUEZ-ESTRADA, CARLOS. Reduced nicotinamide adenine dinucleotide and depolarization in neurons. Am. J. Physiol. 228(4) : 9961001. 1975.-Activity of frog dorsal root ganglion neurons, evoked by dorsal root stimulation under aerobic conditions, produced a change in the level of reduced nicotinamide adenine dinucleotide (NADH) on the surface of this tissue. There was a decrease of NADH (oxidation), followed by an increase of NADH (reduction). Both changes were dependent on previous activity, and a critical time is required in order to observe a similar response. At the frequency and duration of stimulation used here, each stimulus evoked neuron depolarization as shown by recording from single cells. The NADH oxidation occurred in the respiratory chain and it was selectively blocked by Amytal. The NAD reduction was attributed to 3-phosphoglyceraldehyde dehydrogenase activity and it was blocked by iodoacetate. The NADH oxidation and NAD reduction were attributed to respiratory chain activity and aerobic glycolysis, both activated and deactivated by changes of phosphate potential (ATP/ADP + Pi). A low phosphate potential activates the respiratory chain and glycolysis; a high phosphate potential deactivates the respiratory chain and glycolysis. oxidoreduction;

metabolism;

redox;

dinucleotide

phosphate

Facultad

MATERIALS

de Medicina,

AND

METHODS

Dorsal root ganglia of frogs (Rana palmipes spix) were excised and their connective tissue carefully removed under a dissecting microscope. The dorsal root ganglion preparation was placed in a rectangular moist chamber about 65 ml in volume. The chamber was provided with an inlet and outlet for air, stimulation and recording electrodes, and a rubber seal to fit an Ultropak objective (Leitz) or an Epiplan objective (Zeiss). The temperature of dorsal root ganglion could be changed by a stream of warm or cold water under the floor of the chamber. Air humidified by bubbling through water was passed through the chamber at a flow rate of about 10 ml/min. Dorsal root ganglia were moistened during dissection with Ringer solution (NaCl 6.5 g, CaC12 0.2 g, NaHC03 0.2 g, water 1,000 ml, pH 6.8). The peripheral nerve was stimulated with square pulses 0.2 ms in duration. Stimulus voltage twice the threshold of the group A fibers at a frequency of 20 pulses/s was always used; the duration of stimulation varied from 2.5 to 200 s. The Ag-AgCl recording electrodes were placed on the dorsal root. The compound action potential was amplified and displayed on a cathode-ray oscilloscope in order to confirm the presence of conduction. This potential was not reproduced in the figures. The fluorometer was arranged according to the one described by Chance and Legallais (5). The excitation beam passed through an interference filter with a peak transmission at 365 nm (Zeiss) focused onto the preparation. The fluorescence emission passed through a 447-nm (Balzer) or 450-nm graduated. interference filter (Veril S200, Schott and Gen, Mainz) into a photomultiplier (EM1 9558BQ or EM1 6256B). The interference filter was supplemented with a guard filter (Leitz K430 or Zeiss 41) to prevent radiation at the excitation wavelengths from entering the photomultiplier. A neutral filter of known transmittance was used for calibration of fluorescence intensity. Fluorescence variation was expressed as a percentage deviation from the reference level, which was taken as 100 % relative to absence of excitation light. The photomultiplier signal was amplified, displayed on an oscilloscope (Tektronix 502A), and photographed. Temperature variations were less than 0.1 “C. Dorsal root ganglion preparations were incubated in a 5mM solution of Amytal (5-ethyl-5-isoamylbarbiturate) in Ringer solution or 5 mM of sodium iodoacetate in Ringer solution for 5 min, rinsed with Ringer solution, then

potential

REPORT (15) the metabolic activity of neurons in the frog dorsal root ganglion was shown to be related to neuron depolarization. This metabolic activity was observed in aerobic conditions and was characterized by a decrease of reduced nicotinamide adenine dinucleotide (NADH) f o 11owed by an increase of NADH level. An identical metabolic response to a new stimulation was observed after a time interval of about 5 min. In this report fluorometric determinations of NADH in frog dorsal root ganglion neurons were performed before, during, and after peripheral nerve stimulation in aerobic conditions, and intracellular recordings were done in an attempt to more clearly establish a correlation between NADH changes and neuron depolarization. Amytal was used as a blocking agent of the respiratory chain and iodoacetate as an inhibitor of 3-phosphoglyceraldehyde dehydrogenase (3-PGDH) . Nicotinamide adenine dinucleotide is regarded as the hydrogen carrier linked to dehydrogenase activity in the respiratory chain and 3-PGDH. The results show that, after excitation, oxidation of NADH is related to respiratory chain activity and reduction of NAD is related to 3-PGDH activity, and the metabolic response depends on previous activity.

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FIG. 1. Fluorescence variation on dorsal root ganglion after peripheral nerve stimulation. In recordings A, B, and C, taken from same dorsal root ganglion, upper trace is fluorescence recording after peripheral nerve stimulalation and lower trace is temperature recording; retouched stimulus artifact is superimposed on temperature recording. Dashed lines indicate reference fluorescence level. A; fluorescence variations after 5-s period of stimulation before Amvtal treatment. B: fluorescence variations after 5-s period of stimulation after 5 min of Amytal treatment. C: fluorescence variations after 5-s period of stimulation after 30 min of incubation in Ringer solution.

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A stimulation of 5 s duration produced a characteristic fluorescence response, beginning with a decrease of fluorescence (NADH oxidation) and followed by an increase of fluorescence level (NAD reduction). The NADH oxidation starts always ~after the onset of stimulation. The duration of fluorescence variation was about 150 s, as is shown in Fig. IA.

Amytal The fluorescence response in a dorsal root ganglion preparation after 5 min of Amytal treatment was different from the fluorescence response previously recorded. After Amytal treatment NADH oxidation was not observed; only the second deflection (NAD reduction) was observed (Fig. 1B). A recording taken after 20-30 min of incubation in Ringer solution produced the characteristic fluorescence response, similar to that recorded before Amytal treatment (Fig. 1C).

50 set FIG. 2. Fluorescence variation on dorsal root ganglion after peripheral nerve stimulation. In recordings A, B, and C, taken from same dorsal root ganglion, upper trace is fluorescence recording after peripheral nerve stimulation and lower trace is temperature recording; retouched stimulus artifact is superimposed on temperature recording. A: fluorescence variations after 5-s period of stimulation before iodoacetate treatment. B: fluorescence variations after 5-s period of stimulation after 5 min of iodoacetate treatment. C: fluorescence variations after 5-s period of stimulation after 30 min of incubation in Ringer solution.

level observed before stimulation. Later on, at the end of stimulation, a change in the NADH level was not observed, as is shown in Fig. 3. The NADH oxidation starts after the onset of stimulation and the duration of fluorescence variation was similar to that observed after 5 s of stimulation.

Variations of NADH

Iodoacetate The fluorescence response in a dorsal root ganglion preparation after iodoacetate treatment was different from the fluorescence response previously recorded. After iodoacetate treatment NAD reduction was not observed (Fig. ZB). A recording taken after 20-30 min of incubation in Ringer solution produced the characteristic fluorescence response, similar to that recorded before iodoacetate treatment (Fig. 2C). It should be pointed out that the characteristic fluorescence response (NADH oxidation and NAD reduction) was usually not observed after Ringer incubation following iodoacetate treatment.

Variations of NADH Level During Prolonged Stimulation Stimulations characteristic tion. During

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applied during a long period produced the NADH oxidation followed by NAD reducstimulation the fluorescence returned to the

Level after Short Period of Stimulation

Stimulation during NADH variation. In these experiments a second period of stimulation was applied during a) the increasing phase of fluorescence, b) the largest fluorescence, c) the recovering phase of fluorescence, or when d) fluorescence reached the fluorescence level observed before the first stimulation. As shown in Fig. 4, the first stimulation was applied after a resting period of 300 s or more. The second stimulation produced an initial NADH oxidation followed by NAD reduction, but the magnitude of NAD reduction varied. A second stimulation applied 15-20 s after the first stimulation, when the fluorescence was progressively increasing, produced NADH oxidation and NAD reduction, as is seen in Fig. 4B. The area representing the NAD reduction of both responses is larger than twice the area of NAD reduction of the control record (Fig. 4A). A second stimulation applied about 50 s after the first stimulation, when the NADH fluorescence was largest, also


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FIG. 3. Fluorescence varitaions on dorsal root ganglion after peripheral nerve stimulation of 200 s duration. Dashed lines indicate reference fluorescence level.

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FIG. 4. Fluorescence variations on dorsal root ganglion after peripheral nerve stimulation. In recordings from A to E, taken from same dorsal root ganglion, uj$er trace is fluorescence recording after peripheral nerve stimulation and lower trace is temperature recording; retouched stimulus artifact is superimposed on temperature recording. Dashed lines indicate reference fluorescence level. Time interval between stimulation of A and 1st stimulation of B was 300 s. Time interval between 2nd stimulation of B and 1st stimulation of C was 300 s. Time interval between 2nd stimulation of C and 1st stimulation of D was 300 s. Time interval between 2nd stimulation of D and stimulation of E

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produced NADH oxidation followed by NAD reduction, .as is seen in Fig. 4C. However, the area of NAD reduction of both responses is smaller than twice the area of NAD reduction of the control record, but is larger than the area of NAD reduction of control alone (Fig. 4A). A second stimulation applied 75-80 s after the first stimulation, when the fluorescence is decreasing toward the control level, produced NADH oxidation and a small NADH reduction (Fig. 4.@. The area of NAD reduction of both responses is about of the same magnitude as that of the NAD reduction area of the control record (Fig. 4A). A second stimulation applied after the first stimulation, when the level of fluorescence was similar to the level observed previous to the first stimulation, produced the same fluorescence change, but usually the area of NAD reduction of the second fluorescence is smaller. Variations of NADH during periodical stimulation. In these &experiments the first 5-s period of stimulation produced NADH oxidation followed by NAD reduction when it was applied after a resting period of 300 s or more. The second and subsequent periods of stimulation, which were repeated every 50 s, produced NADH oxidation, but NAD reduction progressively diminished until further stimulation had no effect, as shown in Fig. 5, B-J. If the next stimulation period was applied at a longer interval of time, it produced NAD reduction, as shown in Fig. 6B. Stimulation at progressively longer time intervals produced progressively larger NAD reduction, as may be seen in Fig. 6, C-E The

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FIG. 5. Fluorescence variations on dorsal root ganglion after peripheral nerve stimulation at 50-s interval. Continuous recording from A to J. Upper trace is fluorescence recording and lower trace is temperature recording; retouched stimulus artifact is superimposed on temperature recording. Dashed lines indicate reference fluorescence level.

area of NAD reduction increased as the time between stimulation was increased. After a stimulation of 5 s duration repeated every 50 s, when the last stimulation produced NADH oxidation only, it was observed that a shorter or longer stimulation produced NADH oxidation and an unobservable NAD reduction, as is shown in Fig. 7. Notice that the duration of the NADH changes was longer than the stimulation duration. Deflection indicating NADH change increased, reached its largest change, and then declined (Fig. 7, A, B). There was a large deflection after a 5-s stimulation. In a sequence of stimulations repeated every 25-50 s it was observed that the first stimulation produced NADH oxidation followed by NAD reduction. The second and subsequent periods of stimulation produced NADH oxidation and progressively diminished NAD reduction. However, after a few periods of stimulation, another stimulation produced no observable NADH oxidation, as is seen in Fig. 8; after a longer delay stimulation once again produced NADH oxidation. The number of stimulation periods required to eliminate the phase of NADH oxidation varied from preparation to preparation. Intracellular

Potentials

As is shown in Fig. 9A, intracellular recorded at a frequency of 40 pulses/min, by a depolarization with overshoot

action potentials, were characterized and positive after-


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FIG. 6. Fluorescence variation on dorsal root ganglion after peripheral nerve stimulation at increasing time intervals after stimulation at 50-s interval. From A to G, upper trace is fluorescence recording and lower trace is temperature recording; retouched stimulus artifact is superimposed on temperature recording. Dashed lines indicate reference fluorescence level. Time interval between recordings A and B, 100 s ; B andC, 150s;C&dD,ZOOsfDandE, 250s;EandF,300s;FandG, 750s.

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variations on dorsal root ganglion after peripheral nerve stimulation at varied stimulation duration, after repeated stimulation at 50-s interval. Continuous recording from A to ,E. Upper trace is fluorescence recording and lower trace is temperature recording; retouched stimulus artifact is superimposed on temperature recording. Dashed lines indicate reference fluorescence level. A: stimulation of 40 s duration. B: stimulation of 20 s duration. C: stimulation of 10 s duration. D: stimulation of 5 s duration. E: stimulation of 2.5 s duration.

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FIG. 8. Fluorescence variations on dorsal root ganglion after peripheral nerve stimulation. Upper trace is fluorescence recording and lower trace is temperature recording; retouched stimulus artifact is superimDashed lines indicate reference posed on temperature recording. fluorescence level. Last period of stimulation was applied at 50-s interval, others at 25-s interval. In this recording, 1st and 2nd stimulation produced a decrease of fluorescence (oxidation), 3rd and 4th produced no observable fluorescence change, but last stimulation produced a decrease of fluorescence.

potential to each stimulus. Resting membrane potentials ranged from 60 to 80 mV. Intracellular potentials recorded were characteristic of this type of neuron and this species (14). At a frequency of 20 pulses/s each stimulus evoked an

action potential during a stimulation period of 2.5-200 SThe magnitude of the action potential varied from the beginning to the end of the stimulation period. The overshoot and the positive afterpotential decreased progressively, as is shown in a stimulation period of 200 s duration (Fig. 9, B-G). At the end of stimulation the resting and action potential returned to control levels (Fig. 9H). During a 5-s stimulation period, repeated several times every 20 s, each stimulus evoked an action potential. In each period of stimulation the voltage of the action potential decreased progressively, and the overshoot and the positive afterpotential became progressively smaller, as is shown in Fig. 10. Intracellular potentials were not recorded simultaneously with the fluorescence, although they were recorded under the same experimental conditions. DISCUSSION

The fluorescence emission of NADH was elicited by excitation at 365 nm (3). In these experiments on dorsal root ganglion neurons a fluorescence change is considered a variation of NADH level, in agreement with the interpretation given by Chance et al. (3). The fluorescence of dorsal root ganglion neurons showed characteristic changes after a period of stimulation (5 s) and lasted about 150 s. First there was a decrease of fluoresby an increase of fluorescence. These cence, followed fluorescence changes were attributed to an increase of NADH oxidation and NAD reduction, respectively. The fluorescence changes were observed only in aerobic conditions as previously reported (15), and there was a clear association between neuron excitation and aerobic metabolic activity. Oxidation of NADH and reduction of NAD after neuron excitation were not observed after blocking the respiratory chain by anoxia (15), since during anoxia NADH oxidation is blocked in the respiratory chain and NAD is fully reduced (7). It has been known for a long time (11) that the respiratory coefficient is practically unity in nervous tissue in agreement with the oxidation of glucose as the source of energy. The first deflection of the fluorescence response is considered a


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RODRfGUEZ-ESTRADA

FIG. 9. Soma neuron intracellular recording. Recordings A and H show soma neuron depolarization during stimulation at 40 pulses/ min. Recordings from B to G show retouched profile of soma neuron depolarization during 200-s stimulation period at 20 pulses/s.

FIG. 10. Soma neuron intracellular recording. Upper recordmg shows retouched profile of soma neuron depolarization during 5-s stimulation period at 20 pulses/s. Time interval between stimulations was 20 s.

KADH oxidation in the respiratory chain and the second deflection is considered a NAD reduction of NAD-linked dehydrogcnases of metabolic reaction during aerobic glucose utilization. Spectroscopic and biochemical (2, 4) studies have shown that Amytal causes oxidation of flavoproteins and reduction of KAD in the mitochondrial respiratory chain. This indicates a respiratory inhibition by interference with electron transfer between NADH and flavoproteins of the respiratory chain. In this report NADH oxidation was not recorded on dorsal root ganglion neurons treated with Amytal, but an increase of NAD reduction was observed, indicating that oxidation of NADH originated in the respiratory chain and that this metabolic change is blocked by Amytal, just as has been observed in the respiratory chain of isolated mitochondrial preparation (2, 4). The second deflection (NAD reduction) is not blocked by Atnytal at the concentrations used here, indicating that Amytal does not interfere with the transient NAD reduction. Iodoacetate has been considered (17) a rather specific inhibitor of glycolysis and its principal attack is on 3PGDH (9). As a consequence of 3-PGDH inhibition there is a cessation of NAD reduction. In the present study it was observed that after a short period of stimulation of iodoacetate-treated tissue ,NADH.oxidation occurred but there was no NAD reduction. The NADH oxidation was recorded, indicating that iodoacetate does not interfere with the transient KADH oxidation. The above-described metabolic changes of dorsal root neurons induced by excitation were large when the stimulation period (20/s) lasted about 5 s. This stimulation period is approximately the same as that used (12) to evoke large NADH changes in mammalian nonmyelinated nerve fibers. The metabolic changes described in the present study were transitory and lasted about 150 s. Their duration was about the same after a short-term stimulation or after a long stimulation period.

Evidence presented here suggests that NAD reduction originates during tnitochondrial respiration. The respiratory chain is activated (6) by changes in tnitochondrial phosphate potential (ATP/ADP + Pi). Evidence presented here also suggests that NADH reduction originates during aerobic glycolysis. The glycolytic pathway (18) is activated during excitation and evidence had been presented (19) that indicates its control by phosphofructokinase activity. This kinasc is activated and deactivated by the phosphate POtential. After activation of respiration and glycolysis there was phosphorylation and an increase of the phosphate potential, which modifies a newly induced metabolic response. After repeated stimulation NADH oxidation and XAD reduction were reduced in size and finally disappeared, indicating that the phosphate potential had changed by the previous activity and this change modified the metabolic response. A long stimulation clearly shows an attivation and deactivation of the metabolic system. After repeated stimulation NADH oxidation is obser\red when iKAD reduction is not seen, indicating two different sites of glucose metabolism. Both sites have been identified by cooling (12, 15); NAD reduction was not seen at low temperature. In cerebral cortex, where a new acti\-ity may be imposed on a background of activity, NADH oxidation in the respiratory chain (16) has been reported. Variations of metabolic changes induced by excitation after stimulations of variable duration periods cannot be attributed to failure of neuron depolarization; in all stitnulation conditions each stimulus evoked neuron depolarization. The role of calcium was not studied. It has been reported (13) that calcium ions in mammalian nonmyelinated nerves modified NADH oxidation and NAD reduction. The NAD oxidation lagged and NAD reduction was not observed after stimulation in a nerve immersed in a calciumfree solution. As previously reported (15), stimulation in aerobic conditions after a 15-min period of anoxia did not produce


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the NADH changes observed before anoxia, suggesting that anoxia disrupts a possible carrier system. Neuron depolarization in postanoxia conditions is identical to that of preanoxia recording (14) and oxygen utilization of the neurons (14) is not impaired. Changes in light propagation through the cell during the spike process have been reported (8) in squid axon. They were attributed to physical changes of the membrane. The changes in light propagation were reported on birefringence, extrinsic fluorescence, and light scattering. Two components have been described in the changes in light scattering: the first component accompanied the action potential and the second component accompanied the spike aftereffects ( 10). Therefore, membrane physical changes cannot be the origin of the fluorescence observed in the

present study, since the fluorescence variation started after the onset of stimulation, the beginning and end of the second fluorescence varied with stimulation time interval, and the first and second fluorescence variations were not observed after repeated stimulation periods of short duration. I thank Dr. G. P. Cooper and Dr. G. Svaetichin for their valuable suggestions and Angel Cazorla, E. Pfister, and J. R. Perez Hermoso for their excellent technical help. This work was supported by a financial grant from the Fundaci6n Josh Maria Vargas. The intracellular recordings were made possible by a grant from Dr. A. Soto Rivera and R. Valladares. This study has been published in part in abstract form (PhysioZogist 10: 291, 1967). Received

for publication

18 March

1974.

REFERENCES studies of 1. AUBERT, X., B. CHANCE, AND R. D. KEYNES. Optical biochemical events in the electric organ of Electrojhorus electricus. Proc. Roy. Sot., London, Ser. B 160: 21 l-245, 1964. of adenosine diphosphate with the respira2. CHANCE, B. Interaction In : Enzymes: Units of Biological Structure and Function, tory chain. edited by 0. H. Gaebler. New York: Academic, 1956, p. 447-463. 3. CHANCE, B., P. COHEN, F. F. J~BSIS, AND B. SCHOENER. Intracellular oxido-reduction state in vivo. Science 137 : 499-508, 1962. Inhibition of electron and energy 4. CHANCE, B., AND G. HOLLUNGER. transfer in rnitochondria. J. BioZ. Chem. 278: 418-431, 1963. A spectrofluorometer for record5. CHANCE, B., AND V. LEGALLAIS. ing of intracellular oxido-reduction states. IEEE Trans. Bio-Med. Electron. 10 : -40-47, 1963. Respiratory enzymes in oxida6. CHANCE, B., AND G. R. WILLIAMS. IV. Respiratory chain. J. Biol. Chem. 217: tive phosphorylation. 429-438, 1955. 7. CHANCE, B., AND G. R. WILLIAMS. A method for localization of sites for oxidative phosphorylation. Nature 176 : 250-254, 1955. in neuron structure during action potential 8. COHEN, L. B. Change propagation and synaptic transmission. Physiol. Rev. 53 : 373-410, 1973. n-glyceral9. CORI, G. T., M. W. SLEIN, AND C. F. CORI. Crystalline dehyde-3-phosphate dehydrogenase from rabbit muscle. J. Biol. Chem. 173 : 605-618, 1948. B., AND A. L. HODGKIN. The after-effects of 10. FRANKENHAUSER, impulses in the giant nerve fibers of Loligo. J. Physiol., London 13 1: 341-376, 1956.

11. KETY, S. S. Circulation and metabolism of the human brain in health and disease. Arch. J. Med. 8 : 205-217, 1950. 12. LANDOWNE, D., AND J. M. RITCHIE. Optical studies of kinetics of the sodium pump in mammalian non-myelinated nerve fibres. J. Physiol., London 212 : 483-502, 1971. 13. LANDOWNE, D., AND J. M. RITCHIE. On control of glycogenolysis in mammalian nervous tissue by calcium. J. Physiol., London 2 12 : 503-517, 1971. 14. NEGISHI, K., AND G. SVAETICHIN. Effects of anoxia, CO2 and NH3 on S-potential producing cells and on neurons. Pjuegers Arch. 292: 177-205, 1966. C. Fluorometric determinations of NADH2 15. RODRIGUEZ-ESTRADA, levels in the dorsal root ganglion following peripheral nerve stimulation. Brain Res. 6 : 2 17-227, 1967. 16. ROSENTHAL, M., AND F. F. J~BSIS. Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation. J. Neurophysiol. 34 : 750-762, 1971. 17. WEBB, J. L. Enzyme and Metabolic Inhibitors. Iodoacetate and Iodoacetamide. New York: Academic, 1966, vol. 3, p. l-283. 18. WILLIAMSON, J. R., W. Y. CHEUNG, H. S. COLES, AND B. E. HERCZEG. Glycolytic control mechanism. IV. Kinetics of glycolytic intermediate changes during electrical discharge and recovery in the main organ. J. BioZ. Chem. 242: 5112-5118, 1967. 19. WILLIAMSON, J. R., B. E. HERCZEG, H. S. COLES, AND W. Y. CHEUNG. Glycolytic control mechanism. V. Kinetics of high energy phosphate intermediate changes during electrical discharge and recovery in the main organ of Electrophorus electricus. J. BioZ. Chem. 242 : 5119-5124, 1967. ,


The interaction of reduced nicotinamide--adenine dinucleotide phosphate with reduced nicotinamide--adenine dinucleotide--ubiquinone reductase from bovine heart mitochondria.

An enzyme degrading reduced nicotinamide-adenine dinucleotide in Proteus vulgaris.

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

Study of the electrochemical oxidation of reduced nicotinamide adenine dinucleotide.

The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes.

Nicotinamide adenine dinucleotide metabolism in Candida albicans.

In vivo oxidation of reduced nicotinamide-adenine dinucleotide phosphate by paraquat and diquat in rat lung.

Free radical and ionic reaction of bisulfite with reduced nicotinamide adenine dinucleotide and its analogues.

Regulation of citrate synthase activity in methylotrophs by reduced nicotinamide-adenine dinucleotide, adenine nucleotides and 2-oxoglutarate.

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Isolation and properties of reduced nicotinamide adenine dinucleotide phosphate-hepatoredoxin reductase of rabbit liver mitochondria.

Analysis and computer simulation of aerobic oxidation of reduced nicotinamide adenine dinucleotide catalyzed by horseradish peroxidase.

Further characterization of the reduced nicotinamide adenine dinucleotide phosphate: nitrate oxidoreductase in Aspergillus nidulans.

A deactivating conformational change induced by reduced nicotinamide-adenine dinucleotide phosphate in a Neurospora glutamate dehydrogenase.

Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogues.

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Phosphoribulokinase from Nitrobacter winogradskyi: activation by reduced nicotinamide adenine dinucleotide and inhibition by pyridoxal phosphate.

Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide-adenine dinucleotide-binding studies.

The pretectal complex of the rabbit: distribution of acetylcholinesterase and reduced nicotinamide adenine dinucleotide diaphorase activities.

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

Induced transcription-dependent synthesis of mitochondrial reduced nicotinamide-adenine dinucleotide dehydrogenase in Drosophila.

Reduced nicotinamide adenine dinucleotide oxidase involvement in defense against oxygen toxicity of Streptococcus mutans.

The regulation of rat liver tryptophan pyrrolase activity by reduced nicotinamide-adenine dinucleotide (phosphate). Experiments with glucose and nicotinamide.

Evidence for two-step binding of reduced nicotinamide-adenine dinucleotide to aldehyde dehydrogenase.