Energy-linked Transhydrogenase Research Articles

A marriage full of surprises; forty-five years living with glutamate dehydrogenase

Detailed kinetic studies of bovine glutamate dehydrogenase [GDH] from the 1960s revealed complexities that remain to be fully explained. In the absence of heterotropic nucleotide regulators the enzyme follows a random pathway of substrate addition but saturation with ADP enforces a compulsory-order mechanism in which glutamate is the leading substrate. The rate dependence on NAD(P) + concentration is complex and is probably only partly explained by negative binding cooperativity. Bovine GDH eluded successful analysis by crystallographers for 30 years but the final structural solution presented in this symposium at last provides a comprehensible framework for much of the heterotropic regulation, focussing attention on an antenna region in the C-terminal tail, a structure that is missing in the slightly smaller hexameric GDHs of lower organisms. Nonetheless, our studies with one such smaller (clostridial) GDH reveal that even without the antenna the underlying core structure still mediates homotropic cooperativity, and the ability to generate a variety of mutants has made it possible to start to dissect this machinery. In addition, this short personal review discusses a number of unresolved issues such as the significance of phospholipid inhibition and of specific interaction with mRNA, and above all the question of why it is necessary to regulate an enzyme reputedly maintaining its reactants at equilibrium and whether this might be in some way related to its coexistence with an energy-linked transhydrogenase.

Control of the pro-oxidant-dependent calcium release from intact liver mitochondria.

The reduction of molecular oxygen to water provides most of the energy that enables higher organisms to exist. Oxygen reduction is a mixed blessing because incompletely reduced oxygen species are more reactive than molecular oxygen in the ground state and can, when out of control, damage biological molecules. However, incompletely reduced oxygen species may also serve useful functions, as exemplified by their control of mitochondrial Ca(2+) homeostasis, the understanding of which has improved greatly during the last few years. Hydrogen peroxide can stimulate a specific Ca(2+) release pathway from intact mitochondria by oxidizing mitochondrial pyridine nucleotides through the activities of glutathione peroxidase, glutathione reductase, and the energy-linked transhydrogenase. Other pro-oxidants such as menadione, alloxan, or divicine also stimulate the specific Ca(2+) release, because they furnish NAD(+). The specific Ca(2+) release requires for its activation the hydrolysis of intramitochondrial NAD(+) to ADPribose and nicotinamide, and is prevented by inhibitors of NAD(+) hydrolysis and protein monoADPribosylation. Recent experiments reveal that NAD(+) hydrolysis and therefore Ca(2+) release is regulated by vicinal thiols in mitochondria. When reduced or alkylated, the thiols prevent hydrolysis, but when they are cross-linked hydrolysis takes place. Cyclosporine A, which also prevents NAD(+) hydrolysis, acts distal of these vicinal thiols. Since mitochondrial Ca(2+) handling is physiologically relevant, its control by pro-oxidants must be added to the growing list of their useful functions.

Sex differences in the steroidogenic and respiratory electron transport chains in the rat adrenal cortex.

Both steroid 11 beta-hydroxylation and cholesterol side chain cleavage occur in the mitochondria of adrenocortical cells and they require reducing power in the form of NADPH. There are direct sources of NADPH in rat adrenal mitochondria but another potential source of NADPH is the energy-linked transhydrogenase reaction. This suggests that there is a relationship between the steroidogenic and respiratory chains. We have elaborated upon this relationship by exploring the expression of cytochrome c oxidase (CO) and cytochrome P-45011 beta. We have studied the regulation of one mitochondrial-encoded (COII) and one nuclear-encoded (COIV) subunit. Normal, untreated male rats had higher basal levels of activity of CO in adrenal (255%) and liver (144%) mitochondria, compared to normal, untreated female rats. They also had increased COII (300% and 138%) and COIV (300% and 135%). Cytochrome P-45011 beta levels, however, were lower (48%) in adrenal mitochondria of male rats than those of female rats. Androgen treatment of male rats caused an increase in the activity of CO in the mitochondria of the adrenal gland with the levels being 171% of the corresponding controls. This increase in activity paralleled an increase in the levels of COII and COIV in the adrenal as measured by Western analysis. In contrast, adrenal cytochrome P-45011 beta levels were lower (68%). Androgen treatment caused no significant change in the levels of mRNA's for COII and COIV whereas cytochrome P-45011 beta mRNA was significantly lower than normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart

Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart

Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart

Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5′-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label, NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD + and NADP + were inhibitory by themselves under these conditions, and the substrate analogs 5′-AMP and 2′-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2- 3H]-adenosine 5′-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001–1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F 1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close the the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5′-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001–1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.

Inhibition of pro-oxidant-induced mitochondrial pyridine nucleotide hydrolysis and calcium release by 4-hydroxynonenal

Intra- and extra-mitochondrial Ca2+ participates in vital cellular processes. This work investigates the influence of 4-hydroxynonenal (HNE) on pro-oxidant-induced Ca2+ release from rat liver mitochondria. Ca2+ movements across the mitochondrial inner membrane, the pyridine nucleotide redox state and pyridine (nicotinamide) nucleotide hydrolysis were analysed. HNE did not influence Ca2+ uptake by mitochondria, but inhibited in a concentration-dependent manner Ca2+ release induced by t-butylhydroperoxide (tbh). Total inhibition was achieved with about 50 microM-HNE. Ca2+ release induced by the pro-oxidant alloxan was also inhibited by HNE. Oxidation of pyridine nucleotides, induced by tbh through the concerted action of glutathione peroxidase, glutathione reductase and the energy-linked transhydrogenase, was not affected by up to 50 microM-HNE. In contrast, HNE inhibited pyridine nucleotide hydrolysis in a concentration-dependent manner. The data suggest that HNE toxicity may be in part attributed to an impaired intramitochondrial Ca2+ homeostasis.

Energy-linked transhydrogenase. Effects of valinomycin and nigericin on the ATP-driven transhydrogenase reaction catalyzed by reconstituted transhydrogenase-ATPase vesicles.

Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.

Physiological roles of nicotinamide nucleotide transhydrogenase.

From the foregoing considerations, the energy-linked transhydrogenase reaction emerges as a powerful and flexible element in the network of redox and energy interrelationships that integrate mitochondrial and cytosolic metabolism. Its thermodynamic features make it possible for the reaction to respond readily to challenges, either on the side of NADPH utilization or on the side of energy depletion. Yet, the kinetic features are designed to prevent a wasteful input of energy when other sources of reducing equivalents to NADP are available, or to deplete the redox potential of NADPH in other than emergency conditions. By virtue of these characteristics, the energy-linked transhydrogenase can act as an effective buffer system, guarding against an excessive depletion of NADPH, preventing uncontrolled changes in key metabolites associated with NADP-dependent enzymes and calling on the supply of reducing equivalents from NAD-linked substrates only under conditions of high demand for NADPH. At the same time, it can provide an emergency protection against a depletion of energy, especially in situations of anoxia where a supply of reducing equivalents through NADP-linked substrates can be maintained. The flexibility of this design makes it possible that the functions of the energy-linked transhydrogenase vary from one tissue to another and are readily adjustable to different metabolic conditions.

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.

Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria.

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.

The participation of NADP, the transmembrane potential and the energy-linked NAD(P) transhydrogenase in the process of Ca 2+ efflux from rat liver mitochondria

The pyridine nucleotide specificity, the participation of Δψ, and the energy-linked transhydrogenase in the process of Ca 2+ efflux stimulated by the oxidized state of NAD(P) were examined in rat liver mitochondria energized by ascorbate + TMPD. The following observations were made: (i) The Ca 2+ efflux rate is independent of the redox state of mitochondrial NAD, but is at a minimum when mitochondrial NADP is in the reduced state and accelerated several-fold when it is in the oxidized state, (ii) When the redox state of NADP is shifted to a more oxidized state, the steady-state level of Ca 2+ in the medium increased and Δψ decreased in proportion to the mitochondrial NADP + level, (iii) The activity of the energy-linked NAD(P) transhydrogenase seems to be a key element in determining the redox state of NADP and thus of Ca 2+ retention and efflux from mitochondria.

Synthesis of pyrophosphate coupled to the reverse energy-linked transhydrogenase reaction in Rhodospirillum rubrum chromatophores

Chromatophores from the photosynthetic bacterium Rhodospirillum rubrum contain a membrane-bound transhydrogenase catalyzing the transfer of a hydride ion between NADH and NADP +. The reverse reaction, i.e. reduction of NAD + by NADPH, can furnish sufficient energy (Δ \\ ̃ gmH +) to drive the phosphorylation of inorganic orthophosphate (P i) to pyrophosphate (PP i). The rate of pp i synthesis is 50 nmol PP i formed/min per μmol Bchl which is 5% of the rate of light-induced PP i synthesis. PP i synthesis is inhibited by both the H +-PPase inhibitor fluoride and the specific transhydrogenase inhibitor palmitoyl-CoA. The effects of both DCCD and uncouplers on the system provide additional evidence that the Δ \\ ̃ gmH + generated by the reverse transhydrogenase reaction drives PP i synthesis. The rate of PP i synthesis can be partially inhibited by the addition of NADP + a substrate of the forward energy-consuming reaction. The Δ \\ ̃ gmH + generated can also be used to drive ATP synthesis by the H +-ATPase, but at a lower rate than the pp i synthesis.

Inhibition of p-nitroanisole O-demethylation in perfused rat liver by oleate

p-Nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats was rapidly and reversibly inhibited by sodium oleate (0.3 to 0.6mM). Xylitol partially reversed this inhibitory effect. The inhibition was not mediated by a direct effect of oleate on musomal components since concentrations of oleate ranging up to 1.0 mM did not affect p-nitroanisole O-demethylation by isolated musomes. Infusion of 0.6 mM oleate did not alter the measured intracellular NAD +/NADH ratio but did cause a significant increase in the intracellular NADP +/NADPH ratio. A significant decrease in the ATP/ADP ratio was also observed. Oleoyl Co A inhibited p-nitroanisole O-demethylation in musomes ( K i about 30 μM), and both oleoyl Co A and palmitoyl CoA inhibited the energy-linked nicotinamide nucleotide transhydrogenase in submitochondrial particles ( K i about 1 μM). Thus, inhibition of mixed-function oxidation in the intact liver by oleate is most likely mediated by oleoyl CoA. Oleoyl CoA inhibits mixed-function oxidation in the intact liver by acting directly on cytochrome P-450 and by decreasing generation of NADPH via inhibition of key enzymes of the citric acid cycle and the energy-linked transhydrogenase.

Energy-linked nicotinamide nucleotide transhydrogenase. Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography.

Mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was purified by a novel procedure involving fast protein liquid chromatography and characterized with respect to molecular and catalytic properties. The method is reproducible, gives highly pure transhydrogenase as judged by silver staining, and can be modified to produce large amounts of pure transhydrogenase protein suitable for e.g. sequencing and other protein chemical studies. Transhydrogenase purified by fast protein liquid chromatography is reconstitutively active and pumps protons as indicated by an extensive quenching of 9-aminoacridine fluorescence. Under conditions which generate a proton gradient in the absence of a membrane potential the activity of reconstituted transhydrogenase is close to zero indicating a complete and proper incorporation in the membrane and a preferential regulation of the enzyme by a proton gradient rather than a membrane potential. Treatment of reconstituted transhydrogenase with N,N-dicyclohexylcarbodiimide results in an inhibition of proton pump activity without an effect on uncoupled catalytic activity, suggesting that proton translocation and catalytic activities are not obligatory linked or that this agent separates proton pumping from the catalytic activity.

The efficiency of oxidative phosphorylation and the rapid control by thyroid hormone of nicotinamide nucleotide reduction and transhydrogenation in intact rat liver mitochondria.

In confirmation of previous work enhancement of the fluorescence emission of reduced nicotinamide nucleotides in intact rat liver mitochondria was found to depend on incubation conditions. Under standard conditions the enhancement is constant at 4.8-fold in states 3 and 4 and is not altered by thyroidectomy of the animal 6 weeks prior to experiment. The ADP-induced (state 4----state 3----state 4) fluorescence changes are significantly different in intact mitochondria from normal and hypothyroid animals and reflect the decreased rate and efficiency of oxidative phosphorylation after thyroidectomy. Incubation of liver homogenates in vitro for 15 min with 1 microM triiodothyronine before isolating mitochondria significantly restores their ADP response towards normal. Direct addition of hormone to isolated mitochondria was ineffective. Enzymatic measurement of mitochondrial extracts shows that thyroidectomy leads to increases in the contents of NAD(H) by 22% and NADP(H) by 33%. With glutamate as substrate ADP-induced changes in the reduced/oxidized ratio of NAD+ are not significantly altered in hypothyroid preparations. By contrast the NADP+ ratio remains substantially more reduced in state 3 than it does in normal mitochondria. The hypothesis is advanced that the decreased efficiency of hypothyroid preparations in phosphorylating ADP may be the result of increased energy-linked transhydrogenase activity. This is needed to supply NADPH via the glutathione peroxidase for reducing endogenously formed peroxides. Direct reduction of mitochondrial glutathione with dithiothreitol had no substantial effect on ADP/O ratios or on ADP-induced redox cycles in either normal or thyroidectomised preparations. This decisively eliminates the possibility that lowered phosphorylation efficiency is the result of a leak of reducing equivalents via glutathione peroxidase.

Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: Relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity

Formation of excited species such as singlet molecular oxygen during redox cycling (one-electron reduction-oxidation) was detected by low-level chemiluminescence emitted from perfused rat liver and isolated hepatocytes supplemented with the quinone, menadione (vitamin K 3). Chemiluminescence was augmented when the two-electron reduction of the quinone catalyzed by NAD(P)H:quinone reductase was inhibited by dicoumarol, thus underlining the protective function of this enzyme also known as DT-diaphorase. Interference with NADPH supply by inhibition of energy-linked transhydrogenase by rhein or of mitochondrial electron transfer by antimycin A led to a depression in the level of photoemission. Unexpectedly, glutathione depletion of the liver led to a lowering of chemiluminescence elicited by menadione, whereas conversely the depletion of glutathione led to increased chemiluminescence levels when a hydroperoxide was added instead of the quinone. As the GSH conjugate of menadione, 2-methyl-3-glutathionyl-1,4-naphthoquinone, studied with microsomes, was shown also to be capable of redox cycling, we conclude that menadione-induced chemiluminescence of the perfused rat liver does not only arise from menadione itself but from the menadione-GSH conjugate as well. Therefore, the conjugation of the quinone with glutathione is not in itself of protective nature and does not abolish semiquinone formation. A biologically useful aspect of conjugate formation resides in the facilitation of biliary elimination from the liver. Nonenzymatic formation of the conjugate from menadione and GSH in vitro was found to be accompanied by the formation of aggressive oxygen species.

Why are two different types of pyridine nucleotide transhydrogenase found in living organisms?

Two types of pyridine nucleotide transhydrogenases have been reported in living organisms. The energy-linked transhydrogenase is found in mitochondria and in certain heterotrophic and photosynthesizing bacteria, while the non-energy-linked transhydrogenase is found in certain heterotrophic bacteria. The presence of a structurally similar non-energy-linked transhydrogenase in Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens is readily shown in extracts from these bacteria with Western (protein) blotting. This non-energy-linked enzyme is lacking in Escherichia coli, while the presence of a structurally similar energy-linked enzyme in E. coli and in beef heart mitochondria is indicated with the Western blotting technique. Spinach (Spinacia oleracea) lacks the non-energy-linked transhydrogenase occurring in bacteria. The chloroplast enzyme ferredoxin:NADP+ oxidoreductase, which exhibits non-energy-linked transhydrogenase activity, is immunologically distinct from the bacterial transhydrogenases. In order to provide a rationale for the distribution of the two types of pyridine nucleotide transhydrogenases, the steady-state degrees of reduction of the NADP(H) and NAD(H) pools in A. vinelandii (R'NADP(H) and R'NAD(H)) have been measured for cells metabolizing sucrose at a variable oxygen flux (phi O2). It is found that the degree of reduction of the NADP(H) pool is always higher than that of the NAD(H) pool (R'NADP(H) greater than R'NAD(H)) except when phi O2 goes to zero (R'NADP(H) approximately equal to R'NAD(H)). Comparison of these results with literature values indicates that the inequality R'NADP(H) greater than R'NAD(H) is always found in a membrane-enclosed compartment, irrespective of the type of transhydrogenase present. This allows an understanding of the function of the two types of pyridine nucleotide transhydrogenases in vivo. The physiological role of non-energy-linked transhydrogenase is to catalyze the reaction NADPH + NAD+ leads to NADP+ + NADH, that of energy-linked transhydrogenase to catalyze the reaction NADH + NADP+ leads to NADPH + NAD+. Since at equilibrium R'NADP(H) approximately equal to R'NAD(H) the inequality R'NADP(H) greater than R'NAD(H) under steady-state conditions explains the energy requirement in the latter reaction. The dependence of the non-energy-linked transhydrogenase activity of ferredoxin:NADP+ oxidoreductase on R'NADP(H) is compared with that of A, vinelandii transhydrogenase. The results indicate that this activity is unlikely to be of physiological importance in plant chloroplasts.

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD +) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP +, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD + reaction of reconstituted enzyme. NAD + had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H + translocation that was comparable to the decrease in the rate of NADPH → AcPyAD + transhydrogenation. It is concluded that tetranitromethane modification controls the H + translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H +-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.

The energy-linked transhydrogenase in rat liver in relation to the reductive carboxylation of 2-oxoglutarate.

1 The energy-linked transhydrogenase reaction was studied in isolated mitochondria and in submitochondrial particles from rat liver. 2 The rate of the transhydrogenase reaction in intact mitochondria, estimated by measuring the rate of reduction of NADP+ with 3-hydroxybutyrate as hydrogen donor, was about 30 nmol · min−1· mg mitochondrial protein−1 at 25° C. This value was higher than that calculated from the rate of the energy-linked transhydrogenase measured in submitechondrial particles. From the ratio of the activities at 25° C and 37° C in submitochondrial particles and the rate at 25° C in intact mitochondria it was estimated that the rate of the transhydrogenase reaction in intact mitochondria at 37° C is about 80 nmol · min−1· mg protein−1. 3 In intact mitochondria the rate of reductive carboxylation of 2-oxoglutarate with 3-hydroxybutyrate as hydrogen donor was about 20 nmol · min−1· mg protein−1 at 37° C. The main product formed (> 95%) was citrate. 4 The concentration gradient of citrate across the mitochondrial membrane was the same during reductive carboxylation of 2-oxoglutarate as when citrate was added under non-flux conditions, indicating that transport of tricarboxylates does not limit citrate formation. 5 The activity of isocitrate dehydrogenase (NADP), measured in sonicated mitochondria in the direction of reductive carboxylation, was about 20 nmol · min−1· mg protein−1 at 37° C. 6 It is concluded that the rate of formation of citrate by reductive carboxylation of 2-oxoglutarate is limited by the activity of isocitrate dehydrogenase (NADP). However, the rate of citrate formation is sufficiently high to account for the transport of reducing equivalents needed for hydroxylation reactions in the extramitochondrial compartment in liver.

Respiration-linked proton translocation in the moderate thermophileBacillus stearothermophilus

The majority of bacterial respiratory chains which have so far been investigated exhibit ---> H + / O quotients which do not exceed 8 g-ion H +/g-atom O for the oxidation of endogenous substrates or internal NADPH. This value decreases to approximately 6 in organisms which lack an, active, energy-linked nicotinamide nucleotide transhydrogenase or which do not synthesise, or use, a high redox potential cytochrome c, and does not exceed 4 when both of these components are functionally absent [1-4]. The absence of cytochrome c, but not transhydrogenase, also significantly decreases the efficiency of growth [3,4]. These results have generally been interpreted as indicating the presence in the respiratory chain of up to four energy coupling sites, viz. at the level of transhydrogenase (site 0), NADH dehydrogenase (site 1), and the quinone-cytochrome-cytochrome