On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes.

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.     

The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes. Effects of thyroid status and fatty acids

In less than 1 min ouabain maximally inhibits oxygen consumption due to gramicidin-induced ATP turnover by the Na+/K+-ATPase in hepatocytes. Ouabain rapidly inhibits respiration on palmitate or glucose by only 6-10% indicating that the Na+/K+-ATPase plays a minor role in cell ATP turnover. 29% of the extra oxygen consumption of hepatocytes isolated from hyperthyroid rats was inhibited by ouabain showing that the Na+/K+-ATPase is responsible for some but not the majority of the stimulation of respiration induced by thyroid hormone.     

Reducing-equivalent transfer to the mitochondria during gluconeogenesis and ureogenesis in hepatocytes from rats of different thyroid status.

Isolated hepatocytes from hypothyroid, euthyroid and hyperthyroid rats have been employed to investigate the relative importance of reducing-equivalent shuttles for the transfer of hydrogen between cytoplasm and mitochondria during simultaneous ureogenesis and gluconeogenesis. In cells from hypothyroid animals, a 58% depression of glucose formation and 68% reduction in ureogenesis were induced by n-butylmalonate, an inhibitor of the malate shuttle. A more reduced state of the cytoplasmic compartment and a substantial fall in the concentrations of pyruvate, aspartate, alanine and glutamate accompanied this inhibition. Preincubation of cells with n-butylmalonate yielded greater inhibitory effects than observed in the absence of preincubation. The inhibitory effects on gluconeogenesis and ureogenesis were less in cells from euthyroid rats and were very much reduced in the case of glucose synthesis and absent in the case of ureogenesis, in cells from hyperthyroid rats. It is inferred that both the malate-aspartate and alpha-glycerophosphate shuttles may function in the transfer of reducing equivalents from cytoplasm to mitochondria during ureogenesis in hepatocytes. The major inhibition by n-butylmalonate of glucose and urea synthesis in hepatocytes from hypothyroid rats is due to the diminished activity of the alpha-glycerophosphate shuttle in these cells. Moreover, it follows that the NADH arising from the cytoplasmic malate dehydrogenase-catalysed reaction is accessible to both the malate-aspartate shuttle and the alpha-glycerophosphate shuttle.     

Altered relationship between protonmotive force and respiration rate in non-phosphorylating liver mitochondria isolated from rats of different thyroid hormone status

We have determined the relationship between rate of respiration and protonmotive force in oligomycin-inhibited liver mitochondria isolated from euthyroid, hypothyroid and hyperthyroid rats. Respiration rate was titrated with the respiratory-chain inhibitor malonate. At any given respiration rate mitochondria isolated from hypothyroid rats had a protonmotive force greater than mitochondria isolated from euthyroid controls, and mitochondria isolated from hyperthyroid rats had a protonmotive force less than mitochondria isolated from euthyroid controls. In the absence of malonate mitochondrial respiration rate increased in the order hypothyroid less than euthyroid less than hyperthyroid, while protonmotive force increased in the order hyperthyroid less than euthyroid less than hypothyroid. These findings are consistent with a thyroid-hormone-induced increase in the proton conductance of the inner mitochondrial membrane or a decrease in the H+/O ratio of the respiratory chain at any given protonmotive force. Thus the altered proton conductance or H+/O ratio of mitochondria isolated from rats of different thyroid hormone status controls the respiration rate required to balance the backflow of protons across the inner mitochondrial membrane. We discuss the possible relevance of these findings to the control of state 3 and state 4 respiration by thyroid hormone.     

Effect of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes

The effects of the thyroid state on the cytosolic free Ca2+ concentration, [Ca2+]i, of resting and K+-depolarized cardiomyocytes were studied using the fluorescent Ca2+ indicator fura2. The mean resting [Ca2+]i in euthyroid myocytes (89 +/- 8 nM) was not significantly different from that in hyperthyroid myocytes (100 +/- 14 nM). The resting O2-consumption rate was identical for both groups when expressed per mg protein, but a 35% higher value was observed in the hyperthyroid group when expressed per cell on account of the cellular hypertrophy induced by thyroid hormone. Potassium induced depolarization (50 mM [K+]0) raised the level of [Ca2+]i by 50% in both groups. When ATP-coupled respiration was blocked with oligomycin, the 50 mM K+-induced rise in [Ca2+]i was accompanied in both groups by a 40% rise in glycolytic activity as inferred from measurement of lactate production. Ca2+-fluorescence transients were recorded from electrically stimulated myocytes of euthyroid, hyperthyroid and hypothyroid rats. The time taken to reach peak fluorescence (TPL) and that to 50% decay of peak fluorescence (RL0.5) decreased in the direction hypothyroid----hyperthyroid, indicating an increase in Ca2+ fluxes in the same direction. Isoproterenol (1 microM) enhanced the peak Ca2+ fluorescence in electrically stimulated hypothyroid and euthyroid myocytes but not in hyperthyroid myocytes. Both the TPL and RL0.5 were decreased by isoproterenol in euthyroid, but more so in hypothyroid myocytes. None of these parameters were influenced by isoproterenol in the hyperthyroid group. We conclude that (1) thyroid hormone increases neither the O2-consumption rate nor the level of [Ca2+]i of resting cardiomyocytes and (2) the effects of the beta-receptor-agonist isoproterenol on Ca2+ transients of electrically stimulated myocytes, are inversely related to the documented changes in beta-receptor density in heart tissue occurring with alterations in the thyroid state.     

The mechanism of stimulation of respiration by fatty acids in isolated hepatocytes

Addition of fatty acids to isolated hepatocytes raised respiration rate by 92% and raised mitochondrial membrane potential (delta psi m) in situ from 155 to 162 mV suggesting that the increased fuel supply had a greater effect on respiration rate than any increases in processes that consumed mitochondrial protonmotive force (delta p). The relationship between delta psi m and respiration rate was changed by addition of fatty acids or lactate, showing that there was also stimulation of delta p-consuming reactions. In the presence of oligomycin the relationship between delta psi m and respiration rate was unaffected by substrate addition, showing that the kinetics of delta p consumption by the H+ leak across the mitochondrial inner membrane were unchanged. The stimulation of delta p consumers by fatty acids therefore must be in the pathways of ATP synthesis and turnover. Inhibition of several candidate ATP-consuming reactions had little effect on basal or fatty acid-stimulated respiration, and the nature of the ATP turnover reactions in hepatocytes remains speculative. We conclude that fatty acids (and other substrates) stimulate respiration in hepatocytes in two distinct ways. They provide substrate for the electron transport chain, raising delta p and increasing the non-ohmic proton leak across the mitochondrial inner membrane and the rate of oxygen consumption. They also directly stimulate an unidentified delta p-consuming reaction in the cytoplasm. They do not work by uncoupling or by stimulation of intramitochondrial ATP-turnover reactions.     

Effect of thyroid state on rate and sites of H2O2 production in rat skeletal muscle mitochondria

The purpose of this study was to investigate the effects of thyroid state on rates and sites of H(2)O(2) production in rat muscle mitochondria. With Complex I- and Complex II-linked substrates, hypothyroidism decreased and hyperthyroidism increased the rates of O(2) consumption during State 4 and State 3 respiration and the rates of H(2)O(2) release during State 4 respiration. During State 3, the rates of H(2)O(2) release were not affected by thyroid state. However, the mitochondrial capacity to remove H(2)O(2) increased in the transition from hypothyroid to hyperthyroid state, thus suggesting that an increase in H(2)O(2) production rate also occurred in such a transition during State 3 respiration. The observation that mitochondrial coenzyme Q levels and cytochrome oxidase activities are higher in the hyperthyroid and lower in the hypothyroid groups suggests that the modifications of H(2)O(2) production are due to a modulation by thyroid hormone of the mitochondrial content of autoxidizable electron carriers. This idea is supported by measurements of H(2)O(2) release in the presence of respiratory inhibitors. In fact, such measurements indicate that the thyroid state-linked changes in H(2)O(2) production occur at both generator sites of the respiratory chain.     

Interrelationship between drug oxidation ureogenesis and gluconeogenesis in isolated hepatocytes

1. The effect of increased ureogenesis--provoked by NH4Cl and ornithine--on gluconeogenesis and aminopyrine oxidation was studied in isolated hepatocytes prepared from 24 hr starved mice; lactate or fructose was used as gluconeogenic precursor. 2. Increased ureogenesis caused about 40% inhibition both on aminopyrine oxidation and gluconeogenesis when lactate was added as gluconeogenic substrate. 3. On the other hand, only 10% inhibition of aminopyrine oxidation and about 15% inhibition of gluconeogenesis were observed when fructose was used as gluconeogenic precursor. 4. Aminopyrine has been reported to inhibit gluconeogenesis from fructose by 30% and from lactate by 85%. The inhibitory effect of the combined addition of aminopyrine, NH4Cl and ornithine on gluconeogenesis was also dependent on the applied gluconeogenic precursor. 5. The provoked ureogenesis by ammonia and ornithine was not inhibited by aminopyrine. N6, O2-dibutyryl cAMP known to cause an increase of gluconeogenesis a decrease of aminopyrine oxidation enhanced the inhibitory action of increased ureogenesis on aminopyrine oxidation and on gluconeogenesis further. 6. The role of NADPH in the regulation of drug oxidation and ureogenesis is underlined.     

O(2)-sensing signal cascade: clamping of O(2) respiration, reduced ATP utilization, and inducible fumarate respiration

These studies explore the consequences of activating the prolyl hydroxylase (PHD) O(2)-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broad-spectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O(2) consumption and a 300% increase in lactic acid production under basal conditions. This indicates a approximately 75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O(2) consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O(2) consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHI-treated cardiomyocytes. Silencing of hypoxia-inducible factor-1alpha (HIF-1alpha) expression restored the ability of uncoupled PHI-treated myocytes to increase O(2) consumption; however, basal O(2) uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O(2)-sensing pathway is sufficient to actively "clamp" O(2) consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.     

Relationship of energy production to gluconeogenesis in renal cortical tubules.

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 times 10(-3) M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10(-3) M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate-induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.     

On the stimulation of respiration by alpha-adrenergic agonists in perfused rat liver

Interactions between phenylephrine-induced oxygen consumption, lactate and pyruvate output, and urea and glucose production were examined in perfused livers from fed or 48-h-fasted rats. Within 2 min of phenylephrine infusion, oxygen consumption in perfused livers was increased by approximately 40%. Increases in oxygen consumption induced by phenylephrine were essentially abolished in the presence of carboxyatractyloside, whereas those induced by dinitrophenol were still evident. Phenylephrine-induced increases in oxygen consumption were accompanied by enhanced rates of gluconeogenesis and ureogenesis in livers from fed or 48-h-fasted animals. These data indicate that phenylephrine-induced increases in respiration in perfused rat liver may result from an enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.     

Control of respiration and oxidative phosphorylation in isolated rat liver cells

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats. Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply). From the response of respiration to the mitochondrial membrane potential (delta psi M) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate delta psi M, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.     

The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes.

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.     

Effects of thyroid state on H2O2 production by rat heart mitochondria: sites of production with complex I- and complex II-linked substrates.

This work was designed to determine possible effects of altered thyroid states on rates and sites of H 2 O 2 production by rat heart mitochondria. Rates of O 2 consumption and H 2 O 2 release, capacities to remove the peroxide, lipid peroxidation, cytochrome oxidase activities and ubiquinone levels were determined in heart mitochondria from euthyroid, hypothyroid, and hyperthyroid rats. Hypothyroidism decreased, whereas hyperthyroidism increased the rates of O 2 consumption and H 2 O 2 release during both state 4 and state 3 respiration with Complex I- or Complex II-linked substrates. The percentage of O 2 released as H 2 O 2 was not significantly affected by thyroid state. However, the mitochondrial capacity to remove H 2 O 2 increased in the transition from hypothyroid to hyperthyroid state, which indicates that H 2 O 2 production did not modify in proportion to the rate of O 2 consumption. The thyroid-state-linked changes in H 2 O 2 production were well correlated with the levels of hydroperoxides. Rates of H 2 O 2 release in the presence of respiratory inhibitors indicated that changes in the H 2 O 2 production occurred at both sites at which H 2 O 2 was generated in euthyroid state. This result and the observation that ubiquinol levels and cytochrome oxidase activities increase in the transition from hypothyroid to hyperthyroid state suggest that the modifications of H 2 O 2 production are due to a modulation by thyroid hormone of mitochondrial content of autoxidisable electron carriers.     

Cycloheximide: An adrenergic agent

Cycloheximide, a widely used inhibitor of protein synthesis, stimulates glycogenolysis, gluconeogenesis and ureogenesis in isolated rat hepatocytes. The effects of cycloheximide were compared to those of norepinephrine. Both agents, cycloheximide and norepinephrine, produced slight increases in the levels of cyclic AMP (30% increases) which were blocked by propranolol. Interestingly, it was found that the metabolic actions of norepinephrine and cycloheximide (stimulation of glycogenolysis, gluconeogenesis and ureogenesis) were only slightly diminished by the β adrenergic antagonist propranolol but abolished by the selective α₁ adrenergic antagonist prazosin. The ability of cycloheximide to inhibit protein synthesis was not affected by either prazosin or propranolol. It is concluded that the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by cycloheximide in rat hepatocytes, is an effect of the antibiotic independent of its ability to inhibit protein synthesis and that is mediated through activation of α₁ adrenoceptors. The adrenergic activity of cycloheximide should be considered when this drug is used as an inhibitor of protein synthesis.     

Effect of hyperthyroidism and hypothyroidism on lipid and carbohydrate metabolism of the perfused rat liver.

1. Liver from hyper- and hypo-thyroid male fed rats were perfused with whole blood and their metabolism was compared with euthyroid controls. 2. Hyperthyroid livers produced more bile than controls and hypothyroid livers produced less. 3. Glucose output by all livers was similar; glycogen declined only during perfusion of hyperthyroid livers. Lactate uptake increased in hyperthyroid but decreased in hypothyroid livers. These results may be explained by changes in oxidation of carbohydrate rather than in gluconeogenesis. 4. Secretion of triacylglycerol was decreased in hyperthyroid and not changed significantly in hypothyroid livers. 5. Fractional extraction of infused [1-14C]oleate was unaltered. Hyperthyroid livers oxidized more oleate to CO2 and ketone bodies, esterified less and incorporated less into lipoproteins of d less than 1.006. Hypothyroid livers oxidized and esterified oleate to the same extent as controls; their decreased O2 consumption was due to diminished oxidation of other (non-lipid) substrates; 14C-labelled ketone-body formation was increased, but at the expense of 14CO2 production. 6. Lipogenesis (measured with 3H2O) was unaltered in hyperthyroid but was decreased in hypothyroid livers. Incorporation of 3H and 14C into triacylglycerol relative to phospholipid decreased in hyperthyroid and increased in hypothyroid livers. Cholesterol synthesis was similar in all perfusions. 7. During oleate infusion, the cytosolic redox state, as indicated by the perfusate [lactate]/[pyruvate] ratio, was decreased in hyperthyroid and increased in hypothyroid livers. No change in [3-hydroxybutyrate]/[acetoacetate] was detected. 8. The importance of relating the concentration of plasma non-esterified fatty acids to the interpretation of metabolic data obtained under differing thyroid status is emphasized.     

The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis.

The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on phosphorylation coupled with denitrification and aerobic respiration inPseudomonas denitrificans

Formation of ATP during aerobic respiration and denitrification was determined inPseudomonas denitrificans. In the intact cell system, the ATP formation associated with denitrification was almost the same as that associated with aerobic respiration when lactate was used as an electron donor. The ATP formation was inhibited by KCN, NaN₃ and DNP. No phosphate uptake occurred when NH₂OH, DMPD or TMPD was used as an electron donor, although the production of N₂O, N₂ or NO from nitrite was accelerated under anaerobic conditions. In the cell-free system, the ATP formation was also demonstrated using an ATP trapping system and lactate as a substrate. The effects of inhibitors were almost the same as those observed with the intact cells. DMPD or TMPD together with ascorbate promoted the ATP formation during aerobic oxidation by the cell-free system whereas no stimulation of ATP formation was detected during denitrification.