Endogenous Pyridine Nucleotides Research Articles

On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid

Glycyrrhetinic acid (GE), the aglycone of glycyrrhizic acid, a triterpene glycoside which represents one of the main constituents of licorice root, induces an oxidative stress in liver mitochondria responsible for the induction of membrane permeability transition. In fact, GE, by interacting with the mitochondrial respiratory chain, generates hydrogen peroxide which in turn oxidizes critical thiol groups and endogenous pyridine nucleotides leading to the opening of the transition pore. Most likely the reactive group of GE is the carbonyl oxygen in C-11 which, by interacting mainly with a Fe/S centre of mitochondrial complex I, generates an oxygen-centered radical responsible for the pro-oxidant action.

Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel L-lactate translocators.

In the light of the occurrence of L-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize L-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) L-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate L-lactate traffic across the inner mitochondrial membrane. An L-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, L-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of L-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium L-lactate solutions were also monitored. L-Lactate/oxaloacetate and L-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of L-lactate counter-anions outside mitochondria. These L-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in V(max) values and in inhibition and pH profiles, and proved to regulate mitochondrial L-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.

The membrane permeability transition in liver mitochondria of the great green goby Zosterisessor ophiocephalus (Pallas).

Liver mitochondria from the great green goby Zosterisessor ophiocephalus (Pallas) normally exhibit bioenergetic variables (membrane potential 165+/-7 mV; respiratory control ratio 6.6+/-0.4; ADP/O ratio 1.85+/-0.8; means +/- s.e.m., N=6) and activities of physiological transport systems (phosphate/proton symporter, adenine nucleotide antiporter, Ca(2+) electrophoretic uniporter) comparable with those of rat liver mitochondria. When incubated in the presence of Ca(2+) and an inducer agent such as phosphate, these mitochondria undergo a complete collapse of membrane potential accompanied by a large-amplitude swelling of the matrix, influx of sucrose from the incubation medium, release of endogenous Mg(2+) and K(+) (approximately 90% of the total) and of preaccumulated Ca(2+) and oxidation of endogenous pyridine nucleotides. All these phenomena, which are completely eliminated by cyclosporin A and inhibited with different efficacies by Mg(2+) and spermine, demonstrate that the induction of the permeability transition in this type of mitochondria has characteristics similar to those described in rat liver mitochondria. In contrast, the requirement for very high Ca(2+) concentrations (greater than 100 micromol l(-1) for the induction of the permeability transition represents a very important difference that distinguishes this phenomenon in fish and mammalian mitochondria.

Aluminum as an inducer of the mitochondrial permeability transition.

Treatment of rat liver mitochondria with aluminum in the presence of Ca2+ results in large amplitude swelling accompanied by loss of endogenous Mg2+ and K+ and oxidation of endogenous pyridine nucleotides. The presence of cyclosporin A, ADP, bongkrekic acid, N-ethylmaleimide and dithioerythritol prevent these effects, indicating that binding of aluminum to the inner mitochondrial membrane, most likely at the level of adenine nucleotide translocase, correlates with the induction of the membrane permeability transition (MPT). Indeed, aluminum binding promotes such a perturbation at the level of ubiquinol-cytochrome c reductase, which favors the production of reactive oxygen species. These metabolites generate an oxidative stress involving two previously defined sites in equilibrium with the glutathione and pyridine nucleotides pools, the levels of which correlate with the increase in MPT induction. Although the above-described phenomena are typical of MPT, they are not paralleled by other events normally observed in response to treatment with inducers of MPT (e.g., phosphate), such as the collapse of the electrochemical gradient and the release of accumulated Ca2+ and oxidized pyridine nucleotides. Biochemical and ultrastructural observations demonstrate that aluminum induces a pore opening having a conformation intermediate between fully open and closed in a subpopulation of mitochondria. While inorganic phosphate enhances the MPT induced by ruthenium red plus a deenergizing agent, aluminum instead inhibits this phenomenon. This finding suggests the presence of a distinct binding site for aluminum differing from that involved in MPT induction.

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca 2+ efflux without significant release of state-4 respiration, disruption of membrane potential (Δ ψ) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca 2+ efflux associated with a release of state-4 respiration, a disruption of Δ ψ and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.

Okadaic acid-induced decrease in the magnitude and efficacy of the Ca2+ signal in pancreatic beta cells and inhibition of insulin secretion.

1. Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus-response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied. 2. Okadaic acid dose-dependently (IC50 approximately 200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 microM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K+, and its effects were not reversed by activation of protein kinases A or C. 3. The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets. 4. Okadaic acid treatment slightly inhibited voltage-dependent Ca2+ currents in beta cells (perforated patch technique), which diminished the rise in cytoplasmic Ca2+ (fura-2 method) that glucose and high K+ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca2+ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%. 5. Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1-norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter beta cell function. 6. In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca2+ signal in beta cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain beta cell proteins may be involved in the regulation of insulin secretion.

Real time micro-fiberoptic monitoring of endogenous fluorescence in the rat conceptus during hypoxia.

A micro-fiberoptic methodology has been developed for non-invasive, real time measurement of endogenous pyridine nucleotide fluorescence from the surface of the visceral yolk sac (VYS) in intact, viable rat conceptuses. Gestational day (GD) 10-12 conceptuses are maintained in a customized perifusion system, which allows for control of oxygenation, as well as the continuous measurement of pH and oxygen concentration in the effluent perifusate. Miniaturized light guides were constructed by drawing 250 microns ESKA acrylic optical fibers through a stainless steel sheath with a high strength epoxy polymer. A single fiber supplied the excitation signal from a mercury arc lamp at a wavelength of 366 nm. The emission signal was returned via three additional fibers, electronically amplified, processed, and recorded, using a dual channel lamp-compensated fluorometer, optimized for detection of reduced pyridine nucleotides at 455 nm. Endogenous fluorescence in the conceptus was monitored by placing the polished tip of the sensor directly on the surface of the VYS. Oxygen-equilibrated conceptuses, exposed to 100% nitrogen, produced a reproducible biphasic surface fluorescence peak, which returned to baseline levels upon reoxygenation of the perifusate. This biphasic response consisted of an initial rapid rise in fluorescence (phase I), followed by an attenuated rate in fluorescence signal increase (phase II). The hypoxia produced age-dependent rates of fluorescence change during phase I, while phase II remained relatively unchanged throughout GD 10-12. These results demonstrate the ability to monitor endogenous fluorescence, non-invasively and in real time, during the period of organogenesis in the intact rat conceptus and will provide valuable information in studies of embryonic metabolism and response to chemical embryotoxicants.

A mechanism of latent cryoinjury and reparation of mitochondria

Changes in the systems of oxidative phosphorylation and ion transport of rat liver mitochondria have been studied during storage or incubation after freeze-thawing. It has been found that two different processes take place in frozen-thawed mitochondria, one of them tends toward resealing membrane defects and is accompanied by a partial reparation of function; and another one leads to a decrease of the membrane potential, release of K + and Ca 2+ from the matrix, accumulation of lipid peroxides, and uncoupling of oxidative phosphorylation (latent cryoinjury). The latent cryoinjury appears as a result of oxidation of endogenous pyridine nucleotides under conditions of high permeability of the inner membrane and low membrane potential, thus causing activation of the membrane lipid peroxidation and enzyme hydrolysis, leakage of cations, and deenergization of mitochondria. Inhibition of the latent cryoinjury favors the restoration of mitochondrial function after freezethawing

NADP + phosphatase: A novel mitochondrial enzyme

Mitochondria contain a NADP + phosphatase in the matrix space. This is shown by both incubation of mitochondria and subfractions derived thereof with added NADP + and by analysis of endogenous pyridine nucleotides after enzymatic oxidation in Ca 2+-loaded mitochondria. The apparent K M for NADP + is about 1.2 mM. NADPH is not a substrate. The enzyme may be important for modulation of posttranslational modification of macromolecules in mitochondria.

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact cells and isolated protoplasts, by the ability of Ca(2+) and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP(+), SDS(-)) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH.

Metabolic heterogeneity in the perfused rat liver

New methods have been developed to monitor metabolic events non-invasively within periportal and pericentral regions of perfused rat livers. These techniques utilizes two-fiber micro-light guides and miniature oxygen electrodes positioned on identified lobular regions of the perfused liver based on differential pigmentation of periportal and pericentral areas. Two-fiber micro-light guides detect the fluorescence of native and introduced fluors and are used to monitor redox changes of endogenous pyridine nucleotides and the generation of fluorescent products (e.g., 7-hydroxycoumarin) from exogenous substrates. Changes in fluorescence detected with two-fiber micro-light guides are correlated with changes measured with large, multi-fiber light guides and with whole organ rates of metabolism. Subsequently, local rates are estimated. With these techniques, we show that (a) rates of ethanol and acetaldehyde metabolism are similar in periportal and pericentral regions of the liver lobule; (b) mixed-function oxidation predominantes in pericentral regions in livers from phenobarbital-treated rats; (c) rates of sulfation of 7-hydroxycoumarin are greater in periportal than in pericentral hepatocytes; and (d) oxygen uptake is approximately 3-fols greater in periportal than in pericentral areas of the liver lobule.

Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides.

Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca(2+) and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca(2+) is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as beta-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca(2+). Successive cycles of Ca(2+) release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca(2+)-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca(2+), mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport.

Regions of Cerebral Ischemia Located by Pyridine Nucleotide Fluorescence

The fluorescence of the reduced form of the endogenous pyridine nucleotide nicotinamide adenine dinucleotide was used to map regions of ischemia in cat brain. A remarkably microheterogeneous pattern of increased fluorescence resulted from a critical level of incomplete cerebral ischemia. The fluorescence pattern suggests that ischemia occurs initially in microwatershed zones between penetrating cerebral arteries.

Subcellular localization of the F1 and F2 isozymes of horse liver aldehyde dehydrogenase

The subcellular distribution and relative amounts of the two isozymes, F1 and F2, of aldehyde dehydrogenase (EC 1.2.1.3) which were recently purified to homogeneity from horse liver (Eckfeldt, J., et al. (1976) J. Biol. Chem. 251, 236–240) have been investigated. A fresh horse liver homogenate was fractionated on DEAE-cellulose. The results indicate that approximately 60% of the total pH 7.0 acetaldehyde dehydrogenase activity is due to the F1 isozyme and 40% is due to the F2 isozyme. Several horse livers were then fractionated into subcellular components using a differential centrifugation method. Based on the disulfiram (Antabuse) inhibition and the aldehyde concentration dependence of the enzymatic activity, it appears that the disulfiram-sensitive F1 isozyme ( K m acetaldehyde ≅ 70 μ m) is primarily cytosolic and the disulfiram-insensitive F2 isozyme ( K m acetaldehyde ≅ 0.2 μm) is primarily mitochondrial. Fluorescence studies showed that the acetaldehyde dehydrogenase of the intact mitochondria could utilize only the endogenous pyridine nucleotide pool and not externally added NAD. Also, the ethanol dehydrogenase activity was found to be nearly 10 times the total acetaldehyde dehydrogenase activity when assaying a horse liver homogenate at pH 7.0 and with saturating substrates. The significant differences between this work and the results reported in rat liver are discussed with respect to the physiological importance of the cytosolic and mitochondrial aldehyde dehydrogenase during the ethanol oxidation in vivo.

Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in steroid hydroxylation reactions catalyzed by adrenocortical microsomal and mitochondrial cytochrome P 450

This study has investigated the mechanism of steroid hydroxylation in bovine adrenocortical microsomes and mitochondria by employing NaIO 4, NaClO 2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. In the microsomal hydroxylating system, progesterone, 17α-hydroxyprogesterone, and androstenedione were found to act as substrates. Progesterone was chosen as the model substrate and was converted mainly to the 21-hydroxylated derivative in the presence of microsomal fractions fortified with hydroxylating agent. Using saturating levels of hydroxylating agent, NaIO 4 was found to be the most effective in promoting progesterone hydroxylation followed by cumene hydroperoxide, t-butyl hydroperoxide, NADPH, NaClO 2, and pregnenolone 17α-hydroperoxide. Evidence for cytochrome P 450 involvement included a marked inhibition of the activity by substrates and modifiers of cytochrome P 450 and by reagents that convert cytochrome P 450 to cytochrome P 420. Steroid hydroxylation was studied in adrenocortical mitochondria that had been previously depleted of endogenous pyridine nucleotides by aging for 1 h at 30 dgC in a phosphate-supplemented medium. Androstenedione was converted to its respective 6β-, 11β-, 16β-, and 19-hydroxylated derivatives when incubated with aged mitochondrial fractions fortified with hydroxylating agent whereas progesterone was hydroxylated in the 1β-, 6β-, and 15β- positions. These hydroxylations were completely abolished by preheating the mitochondria for 5 min at 95 dgC prior to assay, indicating the enzymic nature of the reactions. Deoxycorticosterone and deoxycortisol were effective substrates for NADPH-dependent enzymic 11β-hydroxylation but were extensively degraded nonenzymically to unidentified products in the presence of NaIO 4 and hydroxylating agents other than NADPH and consequently could not be utilized as substrates in these reactions. Using androstenedione as substrate, NaIO 4 was the most effective hydroxylating agent, followed by cumene hydroperoxide, NaClO 2, t-butyl hydroperoxide, and NADPH. These hydroxylations were inhibited by substrates and modifiers of cytochrome P 450 and by reagents that convert cytochrome P 450 to cytochrome P 420. A mechanism for steroid hydroxylation in adrenocortical microsomes and mitochondria is proposed in which the ferryl ion (compound I) of cytochrome P 450 functions as the common “activated oxygen” species.

Energy metabolism in Saccharomyces cerevisiae: Effect of progressive starvation on respiration and pyridine nucleotide reduction linked to ethanol oxidation in intact cells

The progressive effects of aerobic starvation on endogenous and ethanol-linked respiration and pyridine nucleotide reduction have been studied in the yeast Saccharomyces cerevisiae. Three distinct phases of pyridine nucleotide reduction were observed when ethanol was added to unstarved yeast: an initial phase of rapid reduction and accelerating respiration (A); a steady-state phase of reduction with maximal respiration (B); a final phase of rapid reduction at anaerobiosis (C). During the first 5 hr of aeration, the steady-state Phase B was replaced by a phase of slow pyridine nucleotide reduction, while Phases A and C were unaffected. During this period, both endogenous pyridine nucleotide reduction and endogenous respiration decreased sharply. Between 5 and 22 hr of aeration, the endogenous level of reduced pyridine nucleotide declined further, while endogenous respiration remained unchanged. Concurrently, the extent of the Phase A reduction doubled. The addition of ethanol to aerobic, unstarved yeast stimulated a rapid pyridine nucleotide reduction, with further reduction occurring at anaerobiosis. Under anaerobic conditions, the addition of ethanol to unstarved yeast caused little further reduction of pyridine nucleotide. Two hours of starvation decreased the extent of the endogenously supported anaerobic reduction and correspondingly increased the ethanol-induced reduction. These results suggest that, in unstarved yeast, reducing equivalents derived from ethanol under aerobic conditions and those derived from endogenous carbohydrate under anaerobic conditions have access to the same pool of pyridine nucleotide. With starvation, this pool becomes accessible to ethanol-derived (or ethanol-mobilized) reducing equivalents under anaerobic conditions.

The Respiratory Chain of Plant Mitochondria

Cytochromes b(553), b(557), and b(562) of mung bean (Phaseolus aureus) mitochondria become partially reduced with endogenous substrate on addition of antimycin A to the aerobic mitochondrial suspension. Addition of ATP causes partial reoxidation of the three cytochromes. This partial oxidation by ATP is inhibited by oligomycin and reversed by uncoupler. Ubiquinone does not appear to act as electron acceptor for the oxidation reaction, but a nonfluorescent flavoprotein, or possibly ironsulfur protein, component does appear to act as acceptor. This is consistent with reverse electron transport driven by ATP across the first site of energy conservation of the respiratory chain. Endogenous pyridine nucleotide and the fluorescent flavoprotein with E(m7.2) = -155mv (midpoint potential at pH 7.2, referred to normal hydrogen electrode) in uncoupled mitochondria become reduced in anaerobiosis attained by oxidation of succinate in the absence of respiratory inhibitors of the cytochrome chain, provided that Pi and ATP are present. Under these same conditions, cytochrome b(557) is completely reduced but cytochrome b(562) remains nearly completely oxidized. There is no equilibration across the first site of energy conservation between the carriers on the low potential side and cytochrome b(562) with E(m7.2) = -77mv on the high potential side. It is concluded that cytochrome b(562) is not a part of the main sequence of electron transport carriers of the mitochondrial respiratory chain of plants; it can participate in redox reactions with the respiratory chain in coupled mitochondria but not in uncoupled mitochondria unless antimycin A is present.

Calcium and pyridine nucleotide interaction in mitochondrial membranes

The interaction of NADH with divalent cations was studied in model systems and in isolated rat liver mitochondria. The addition of Ca 2+ to solutions of NADH in methanol, but not in water, produces a large increase in fluorescence intensity and a shift in the emission spectrum to a shorter wavelength. The ratio of fluorescence intensity in the presence of Ca 2+ with respect to that in the absence of Ca 2+ is 1 in aqueous and 2.4 in methanol solutions. Other di- or trivalent cations produce a similar enhancement of NADH fluorescence in nonpolar solutions, except for Mn 2+ which quenches the fluorescence of NADH. The energy-dependent accumulation of Ca 2+ in mitochondria increases the NADH fluorescence in the presence of rotenone. This effect was absent when Mn 2+ was previously accumulated by mitochondria. With the NADH-linked substrates, but not with succinate and rotenone, the accumulation of large amounts of Ca 2+ in mitochondria causes a depression of respiration and a release of the accumulated Ca 2+. The inhibition of the respiration is accompanied by an increased permeability and a depletion of the endogenous pyridine nucleotides. Both effects were not directly related to the concomitant mitochondrial swelling. On the basis of this data, the formation of a complex between calcium and NADH in the nonpolar region of the mitochondrial membrane is proposed. The physiological implications of this complex are discussed.

The Respiratory Chain of Plant Mitochondria

The cytochromes c of mung bean (Phaseolus aureus) mitochondria become reduced when sulfide, a cytochrome oxidase inhibitor free from uncoupling side effects, is added to the aerobic mitochondrial suspension in the absence of added substrate. The cytochromes b remain largely oxidized. Subsequent addition of ATP results in partial oxidation of the cytochromes c and partial reduction of the cytochromes b due to ATP-driven reverse electron transport through the second site of energy conservation, or coupling site, of the respiratory chain. Cytochrome a is also oxidized under these conditions, but there is no concomitant reduction of the flavoprotein components, of ubiquinone, or of endogenous pyridine nucleotide. The reaction is abolished by oligomycin. The reducing equivalents transported from the cytochromes c and a in ATP-driven reverse electron transport are about 2-fold greater than those which appear in the cytochromes b. It is suggested that the equivalents not accounted for are present in a coupling site enzyme at the second site of energy conservation which interacts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbance changes with redox state over the wavelength range examined. With succinate present, reverse electron transport can be demonstrated at both coupling sites in both the aerobic steady state and in anaerobiosis. ATP-driven reverse electron transport in anaerobiosis maintains cytochrome a 30% oxidized while endogenous pyridine nucleotide is 50% reduced.When mung bean mitochondria, oxidizing succinate in the presence of sulfide through the alternate, cyanide- and sulfide-insensitive terminal oxidase, become anaerobic, cytochrome b(557), which has remained largely oxidized, becomes slowly reduced. The slow reduction is observed in coupled, energized mitochondria and in uncoupled mitochondria; the time course parallels the reduction of cytochrome a(3) under the same conditions. It appears that sulfide-liganded, oxidized cytochrome a(3) may be in close enough proximity to cytochrome b(557) in the membrane to inhibit the reduction of the latter.

NAD +-induced phosphate acceptor specificity in submitochondrial systems

The nonspecific phosphorylation reaction in submitochondrial systems obtained by sonication became much more specific in the presence of pyridine nucleotides. Phosphorylation of IDP was inhibited by increasing concentrations of NAD +, an inhibition which became biphasic upon the addition of hexokinase (EC 2.7.1.1), whereas phosphorylation of ADP was totally unaffected. The inhibition by NAD + was of a noncompetitive type with K i = 1.5 · 10 −4 M. The nicotinamide part of the molecule was immediately responsible for the inhibitory effect. Although rotenone or antimycin A interfered with the specificity promoted by NAD +, the pyridine nucleotide effect was not due to the introduction of an energy-competing NAD + reduction. No evidence was found of an initiated selective nucleoside triphosphatase reaction, so the regulation was considered to take place at a primary event of the energy conservation mechanism and possibly exerted by an allosteric change. It is suggested that endogenous pyridine nucleotides which are lost upon sonic disintegration of mitochondria may constitute a factor for regulation of the nucleoside specificity during oxidative phosphorylation.