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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 biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.

NAD+ reduction catalyzed by transhydrogenase (EC 1.6.1.1) from E. coli membrane particles at the expense of NADPH oxidation is coupled with phenyldicarbaundecaborate (PCB-) absorption by the particles. This process is inhibited by oxidative phosphorylation protonophorous uncouplers and by equilibration of concentrations of the substrates and products of the transhydrogenase reaction. Elimination of the water-soluble part of membrane ATPase results in the inhibition of PCB- absorption at the expense of the transhydrogenase reaction energy. Treatment of the particles by dicyclohexyl carbodiimide increases the transhydrogenase-coupled absorption of PCB-. The transhydrogenase-induced increase of pPCB in the suspension of particles is directly correlated with the ratio of ([NADPH].[NAD+])/([NADP+].[NADH]). When this value is equal to 1, no energy-dependent increase of pPCB was observed. NADP+ reduction at the expense of NADH oxidation leads to a decrease in the amount of PCB- absorbed by the particles at the expense of ATP hydrolysis energy. The experimental data suggest that NADPH oxidation in the course of the transhydrogenase reaction is coupled with the formation of a membrane potential with a positive charge localized inside the particles.

The roles of NAD- Specific Isocitrate Dehydrogenase (NAD- IDH) (EC 1.1.1.4), NADP- Specific Isocitrate Dehydrogenase (NADP- IDH) (EC 1.1.1.42) and Piridin Nucleotide Transhydrogenase (Transhydrogenase) (EC 1.6.1.1) in the mitochondrial oxidation of isocitrate through the respiratory chain in conditions of normal and increased energy requirements have been studied in submitochondrial particles isolated from healthy and ischemic dog hearts. The activities of both, NAD- IDH and NADP- IDH were increased in conditions of anoxia, while Transhydrogenase remained unchanged. The results obtained showed that the mitochondrial oxidation of isocitrate in dog myocardium occurs mainly through the NAD- IDH pathway in normoxic and anoxic conditions.

Dicyclohexylcarbodiimide (DCCD) inhibits the reduction of oxidized 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH catalyzed by purified and bovine heart submitochondrial particle transhydrogenase. Kinetic studies demonstrate that the modification of 1 residue results in complete inactivation. Both transhydrogenase preparations were labeled with [14C]DCCD. Labeling of the purified enzyme was time-dependent and paralleled the extent of inhibition. The incorporation of approximately 1 mol of [14C]DCCD/monomer resulted in complete inactivation of the enzyme. At longer preincubation times or at higher DCCD concentrations, more than 1 mol of DCCD reacted and cross-linked dimers of transhydrogenase were formed. The effect of substrates on DCCD inactivation was investigated. AcPyAD+ provided no protection, and NADH gave partial protection in submitochondrial particles, but not of the purified enzyme. NADPH and NADP+ stimulated inhibition. These results indicate that DCCD modification occurs outside the active site. In experiments with transhydrogenase reconstituted into K+-loaded phosphatidylcholine liposomes, DCCD inhibited the rate of H+ uptake into the vesicles to a significantly greater extent than transhydrogenation. The incorporation of approximately 1 mol of DCCD/mol of transhydrogenase monomer completely inhibited H+ translocation, whereas complete inactivation of hydride ion transfer accompanied the incorporation of approximately 2 mol of DCCD. These results indicate that DCCD modified transhydrogenase outside the active site, possibly in a putative H+-binding domain that functions to translocate protons across the membrane by a pump rather than by a loop mechanism.

Exposure of freshwater fish to saline conditions brings about somewhat drastic changes in the mitochondrial energy metabolism. These include abolition of oxidative phosphorylation, ATP-induced contraction of swollen mitochondria and transhydrogenase activity. On the other hand the endogenous calcium levels and protein synthetic capacity are elevated. In vitro protein synthesis by mitochondria from freshwater and stressed fish shows qualitative and quantitative variations. Effluxing the excess calcium by treatment with NaCl or inhibiting the protein synthesis by chloramphenicol in stressed mitochondria restores almost all the functions. It is proposed that the energy potential formed by the mitochondrial membrane is channelized to perform different functions and that the ratio of channelization can be altered to suit the needs of the cell.

Reconstitution of homogeneous bovine heart mitochondrial transhydrogenase into phosphatidylcholine liposomes results in a greater than 80% inhibition of NADPH leads to 3-acetylpyridine adenine dinucleotide (AcPyAD+) transhydrogenation. This coupled rate is stimulated 5-fold by addition of protonophore to the rate observed with unreconstituted enzyme. In the absence of uncoupler, addition of low concentrations (10 microM) of NADH promotes by about 3-fold the rate of reduction of AcPyAD+ in the presence of NADPH. In the absence of NADPH, no reduction of AcPyAD+ by NADH occurs. Addition of valinomycin to K+-loaded proteoliposomes stimulated NADPH leads to AcPyAD+ transhydrogenation to the uncoupled rate and allowed the uptake of protons from the medium. In the absence of valinomycin, the rapid reduction of AcPyAD+ seen on addition of NADH was not coupled to proton translocation. In the presence of valinomycin, NADH addition neither equilibrated protons across the liposomal membrane nor affected the stoichiometry of proton uptake to hydride ion transfer (H+:H- ratio). Addition of NADH to proteoliposomes in the presence of AcPyAD+ and NADPH resulted in an initial rapid formation of reduced 3-acetylpyridine adenine dinucleotide comparable to the amount of NADH added. Thereafter, the rate of AcPyAD+ reduction returned to that seen in the absence of NADH. These results suggest that the NADH requirement for AcPyAD+ reduction is stoichiometric rather than catalytic. Stereospecificity studies demonstrate that reconstituted transhydrogenase catalyzes a direct transfer of a hydride ion equivalent from the 4A locus of NADH to the 4A locus of the NADH product in the presence, but not in the absence, of NADPH. It is proposed that NADH leads to NAD+ transhydrogenation represents a partial reaction of NADPH leads to NAD+ transhydrogenation which involves the participation of a reduced-enzyme intermediate.

1. Purified and reconstituted nicotinamide nucleotide transhydrogenase from beef heart mitochondria was investigated with respect to kinetic and regulatory properties in uncoupled and coupled liposomes. 2. Double reciprocal plots of initial velocities for the reduction of NAD+ by NADPH versus substrate concentrations were convergent and intersecting on or close to the abscissa, indicating a ternary complex mechanism. The effect of site-specific inhibitors indicates that the order of addition of the substrates to the enzyme is random. 3. Reconstituted transhydrogenase uncoupled by FCCP reveals kinetic properties that are indicative of energization, i.e. an increased and decreased affinity for NADP+ and NAD+, respectively, suggesting that reconstituted transhydrogenase is maintained in an activated conformation. An increased extent of coupling causes a progressively increasing change in the same direction. These results suggest that the uncoupler-dependent enhancement of the rate of reduction of NAD+ by NADPH is due to a decreased Km for NAD+. 4. Reconstituted transhydrogenase catalyzes a transhydrogenation between NADH and 3-acetylpyridine adenine dinucleotide (oxidized) in the presence of NADPH. Reconstituted transhydrogenase also also catalyzes the reduction of thio-NADP+ by NADPH in the presence of NADH. Both reactions are concluded to occur indirectly through the generation of NADP+ and NAD+, respectively, and not directly through a reduced enzyme intermediate. 5. A proton pump mechanism is proposed for transhydrogenase which involves a dimeric form of the enzyme where the two subunits are alternating in proton pumping.

When a freshwater fish, Tilapia mossambica (now renamed Sarotherodon mossambicus) is exposed to a salinity stress, extensive changes are noted in the properties of the mitochondria isolated from the gill tissue. Our efforts were directed toward ascertaining the response of the gill mitochondria to an ion-osmotic shock to which the organism is exposed. Notable among them are the loss of ability to phosphorylate externally added ADP, decreased 45Ca++ uptake, lower transhydrogenase levels, and nonresponse to ATP + Mg++ for contraction. During this period there is a large influx of calcium into the mitochondria. Continued exposure to the stress situation reverses virtually all the changes to the freshwater levels.

1 Purified transhydrogenase from Azotobacter vinelandii forms very stable polymers at neutral pH. These show little tendency to dissociate upon dilution but can be disoociated by raising the pH. 2 The stability of transhydrogenase polymers towards dilution can be demonstrated with enzyme that is in part covalently bound to Sepharose. Non-covalently bound subunits are kept bound upon elution at pH 7.5 and can only be released by raising the pH of the eluting buffer (pH 9.0). The remaining dissociated immobilized polymers can be reconstituted by adding free transhydrogenase at a lower pH (7.5–8.5). Reconstitution of dissociated immobilized A. vinelandii polymers can also be achieved with enzyme from Pseudomonas aeruginosa, which demonstrates the close structural relationship between these two enzymes. 3 Differential centrifugation of cell suspensions lysed with the osmotic shock method in the presence of EDTA shows transhydrogenase to be present largely as the non-polymerized octamer (S20,w= 15 S). However, the distribution of the enzyme activity changes markedly when divalent cations (MgCl2, CaCl2) are included in the lysis buffer: up to 65% of the transhydrogenase activity then co-sediments with membranes and nucleic acids (30 min, 20000 × g). Nuclease treatment leads to disappearance of the rapidly sedimenting transhydrogenase fraction. These results are incompatible with a reversible divalent-cation-mediated self-association of the enzyme in vivo. We conclude, on the contrary, that the transhydrogenase octamer is partly immobilized in vivo by divalent-cation-mediated interactions with cell constituents (e.g. nucleic acids) preventing its self-association. 4 The reduction of NAD+ by NADPH catalyzed by cell-free extract transhydrogenase is not influenced by phosphate ions, while the reverse reaction is completely inhibited by 5 mM phosphate. This inhibition is released by adding excess divalent cations (10 mM CaCl2 or MgCl2). Since other metabolites (adenosine, 5′AMP, ADP, ATP) inhibit both activities more or less to the same extent it follows that the antagonistic action of phosphate and divalent cations is of primary importance in regulating the direction of intracellular transhydrogenation in A. vinelandii. 5 The existence of binding sites for these ions on the transhydrogenase subunit is indicated by studies on the inhibition of cell-free extract transhydrogenase with the divalent thiol reagent p-aminophenylarsenoxide. The rate of reaction is slowed down by magnesium and accelerated by phosphate ions.

The relationship between oxidized nicotinamide adenine dinucleotide (phosphate) [NAD(P)+] transhydrogenase (EC 1.6.1.1) and NAD(P)+ glutamate dehydrogenase in several enteric bacteria which differ slightly in their regulation of nitrogen metabolism was studied. Escherichia coli strain K-12 was grown on glucose and various concentrations of NH4Cl as the sole nitrogen source. In the range of 0.5 to 20 mM NH4Cl, the energy-independent transhydrogenase increased two to threefold. Comparable changes occurred in NAD(P)+-linked glutamate dehydrogenase. NH4Cl concentrations of 20 to 60 mM resulted in relatively constant specific activities for both enzymes. Higher exogenous NH4Cl, however, led to a decline in both activities. Isocitrate dehydrogenase, another potential source of cellular NADPH, was insensitive to NH4Cl limitation. Similar studies in the presence of glutamate and different exogenous NH4Cl concentrations again showed concerted effects on both enzymes. Growth on glutamate as the sole nitrogen source led to severe repression of both transhydrogenase and glutamate dehydrogenase. In Salmonella typhimurium, both enzymes were unaffected by limiting NH4Cl or growth on glutamate as the sole nitrogen source. Both were, however, repressed by growth on aspartate, a potential source of cellular glutamate. Coordinate changes in glutamate dehydrogenase and transhydrogenase were also evident in Klebsiella aerogenes, particularly under conditions in which glutamate dehydrogenase was regulated inversely to glutamate synthetase. Coordinate changes in glutamate dehydrogenase and transhydrogenase in enteric bacteria are discussed in terms of the possible involvement of the latter enzyme as a direct source of NADPH in the ammonia assimilation system.

N,N'-di-n-propyl-, N,N'di-n-butyl-, N,N'-di-n-pentyl-, N,N'di-n-hexyl-, N,N'di-n-octoyl, N,N'-dibenzhydryl-, and N,N'-dibenzhydrylcarbodiimides were synthesized. They were all effective inhibitors (2 nmoles carbodiimide per milligram protein) of the ATP-driven reduction of NAD by succinate and the ATP-driven transhydrogenase activities catalyzed by beef heart submitochondrial particles (SMP). They had no effect on the nonenergy-linked transhydrogenase and stimulated the succinate-driven aerobic transhydrogenase activity of beef heart SMP. It was concluded that they exert their effects by reacting with the N,N'-dicyclohexylcarbodiimide-binding protein. Water-soluble carbodiimides were not effective inhibitors.

The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD+ were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)--an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276-287)--when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD+ required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the delta mu H+. In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al.

Respiration- and ATP-dependent transhydrogenation of NADP+ by NADH in everted membrane vesicles from Escherichia coli is inhibited by nigericin but is relatively insensitive to valinomycin. The sensitivity to nigericin is enhanced 30-fold in the presence of valinomycin. It is concluded that both the transmembrane pH difference and the membrane potential constitute the driving force for energy-dependent transhydrogenation.Respiration- and ATP-dependent transhydrogenation are inhibited by tributyltin chloride. Although effects on the energization system have not been excluded, the inhibitor appears to react with a sulfhydryl group on the transhydrogenase enzyme. This inhibition is not dependent on the presence of a permeant anion and can be reversed by mono- and particularly di-thiol compounds. The transhydrogenase is also inhibited by 5,5′-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid, and Zn2+, but these reagents are less effective than tributyltin chloride. Energy-independent transhydrogenation is inhibited at high concentrations (20 mM) of cysteine. The reason for this is unknown.

A mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase (EC 1.6.1.1) was isolated by assaying activity in clones of cells mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. The mutant is missing both energy-independent and energy-dependent transhydrogenase, but has normal NADH dehydrogenase and ATPase activities. Compared to the parental strain, the mutant has normal growth rates with glucose, glycerol, or succinate aerobically and with glucose or glycerol plus fumarate anaerobically. The aerobic growth yield with limiting glucose concentrations is also normal. These growth properties indicate that the enzyme is not an essential source of NADPH or ATP in vivo.

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the mitochondrial energy-linked nicotinamidenucleotide transhydrogenase (TH). Our studies [Phelps, D.C.,Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221; Phelps, D.C.,Hatefi, Y. (1984) Biochemistry 23, 4475-4480] suggested that the inhibition site of DCCD is near the NAD(H) binding site, because NAD(H) and competitive inhibitors protected TH against inhibition by DCCD and, unlike the unmodified TH, the DCCD-modified TH did not bind to NAD-agarose. Others [Pennington, R.M.,Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] could not demonstrate protection by NADH, obtained data indicating DCCD inhibits proton translocation by TH much more than hydride ion transfer from NADPH to 3-acetylpyridine adenine dinucleotide (AcPyAD), and concluded that DCCD modifies an essential residue in the proton channel of TH. The present studies show that N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inhibits TH. The inhibition is pseudo first order at several EEDQ concentrations, and the reaction order with respect to [EEDQ] is unity, suggesting that inhibition involves the interaction of one molecule of EEDQ with one active unit of TH. The EEDQ-modified TH reacts covalently with [3H]aniline, suggesting that the residue modified by EEDQ is a carboxyl group. More significantly, it has been shown that the absorbance change of oxonol VI at 630 minus 603 nm is a reliable reporter of TH-induced membrane potential formation in submitochondrial particles and that TH-catalyzed hydride ion transfer from NADPH to AcPyAD and the membrane potential induced by this reaction are inhibited in parallel by either DCCD or EEDQ.

The mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to minimal assembly of the purified enzyme and of the enzyme in the mitochondrial inner membrane. Studies of the hydrodynamic properties of the purified enzyme in the presence of 0.3% Triton X-100 allowed determination of the Stokes radius, sedimentation constant, partial specific volume, frictional ratio, and molecular weight. Under these conditions transhydrogenase existed as an inactive monomer, suggesting that monomerization may be accompanied by inactivation. Radiation inactivation was used to determine the functional molecular size of purified detergent-dispersed transhydrogenase and transhydrogenase in beef heart submitochondrial particles. Under these conditions the catalytic activity of both the purified and the membrane-bound enzyme was found to be catalyzed by a dimeric form of the enzyme. These results suggest for the first time that the minimal functional assembly of detergent-dispersed as well as membrane-bound transhydrogenase is a dimer, which is not functionally associated with, for example, complex I or ATPase. In addition, the results are consistent with the possibility that the two subunits of transhydrogenase are catalytically active in an alternating fashion according to a previously proposed half-of-the-sites reactivity model.

The membrane vesicles (proteoliposomes) have been reconstituted from soya bean phospholipids and mitochondrial transhydrogenase (EC 1.6.1.1) by a self-assembly procedure. Palmitoyl-CoA and Mg2+ inhibit the rate of NAD+ reduction by NADPH in these proteoliposomes as well as in these submitochondrial particles. After solubilization of transhydrogenase from submitochondrial particles membranes the specific activity of the enzyme decrease and then again increases (more than 4.5 times) after its incorporation into the proteoliposomal membranes. An addition of potassium cholate to the proteoliposomal suspension further increases the rate of the direct transhydrogenase reaction (by 35--40%) due to the function of the oppositely oriented molecules of transhydrogenase. The Michaelis constants for NAD+ and NADPH for transhydrogenase proteoliposomes are 27 microM and 30 microM, respectively. These data are practically coincident with the results obtained for submitochondrial particles (21 and 33 microM, respectively). Thus, the incorporation of transhydrogenase into proteoliposomal membranes results in reconstitution of both electrogenic and the most essential kinetic properties of the enzyme.

The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin.

The behavior of the principal NADPH-consuming pathways, fatty acid synthesis (-ATP-citrate lyase), transhydrogenase, glutamate dehydrogenase, glutathione reductase, NADPH-cytochrome c reductase and mitochondrial and cytoplasmic thioredoxin reductases were studied during the development of the heart, brain and liver of the rat. The liver is the tissue with highest NADPH-consuming activities. Transhydrogenase activity is highest in the heart; it undergoes two steep increases of activity, one at birth and another between 15 and 25 days. Liver cytoplasmic glutathione reductase activity increases mainly at birth. The activities in heart and brain are lower and the brain enzyme activity slightly increases throughout development. Liver NADPH-cytochrome c reductase activity greatly increases at weaning. The heart activity is the lowest, no changes taking place in the developmental period. Heart mitochondrial and brain cytoplasmic thioredoxin reductases undergo increases in activity at birth. The ratios of NADPH-producing/NADPH-consuming activities are constant in all tissues studied, and oscillate between congruent to 4 in fetuses to congruent to 2 in adults, the only exception being the adult heart ratio (congruent to 23). This otherwise constancy confirms that both processes are closely correlated.

1. The activities of enzymes participating in the regeneration of reduced glutathione (GSH), and their subcellular distribution were studied in cultured rat adrenal cells. 2. It has previously been shown that the adrenocorticolytic agent 7-hydroxymethyl-12-methylbenz[a]anthracene (7-hydroxymethyl-12-MBA) causes a drastic and selective oxidation of mitochondrial GSH in rat adrenal cells. Treatment of the adrenal cells with 7-hydroxymethyl-12-MBA, resulted in a minor decrease in the content of cytochrome c oxidase, nicotinamide nucleotide transhydrogenase, isocitrate dehydrogenase and cytosolic GSH reductase, whereas the activity of lactate dehydrogenase and citrate synthase was unaffected. None of these effects were considered to be responsible for the massive oxidation of mitochondrial GSH induced by 7-hydroxymethyl-12-MBA. 3. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) was used to obtain rat adrenal cells cultures with inactivated cytosolic and mitochondrial GSH reductase. The oxidation of mitochondrial GSH, induced by 7-hydroxymethyl-12-MBA, was not dramatically enhanced by the inactivation of GSH reductase, indicating that this enzyme was not rate-limiting in the regeneration of GSH. 4. Fractionation of rat adrenal cells with increasing concentrations of digitonin resulted in an earlier release of citrate synthase in cells treated with 7-hydroxymethyl-12-MBA compared with controls. These results may indicate damage to mitochondrial membranes as a result of 7-hydroxymethyl-12-MBA treatment.

The 2,4-dinitrophenyl derivative of dephospho-CoA and the 7-nitrobenzofurazan-4-yl derivative of CoA are competitive inhibitors (Ki 3 microM and 2.6 microM respectively) of mitochondrial transhydrogenase with regard to NAD+ and NADPH respectively. The 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA is a competitive inhibitor with regard to both transhydrogenase substrates with the same Ki equal to 0.3 microM. The pattern of transhydrogenase inhibition with the 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA indicates that one molecule of the inhibitor binds simultaneously to both the NADP(H) and the NAD(H) binding sites of the enzyme. This result is evidence of the short distance between the NADP(H) and the NAD(H) binding sites.

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.

Modification of mitochondrial nicotinamide nucleotide transhydrogenase (NADPH: NAD+ oxidoreductase, EC 1.6.1.1) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), followed by measurement of the absorption or fluorescence of the transhydrogenase-NBD adducts, resulted in a biphasic labelling of approx. 4-6 sulfhydryls, presumably cysteine residues. Of these 1-2 (27%) were fast-reacting and 3-4 (73%) slow-reacting sulfhydryls. In the presence of substrates, e.g., NADPH, the labelling was monophasic and all sulfhydryls were fast-reacting, suggesting that the modified sulfhydryls are predominantly localized peripheral to the NAD(P)(H)-binding sites. The rates of modification allowed the calculation of the rate constants for each phase of the labelling. Both in the absence and in the presence of a substrate, e.g., NADPH, the extent of labelling essentially parallelled the inhibition of transhydrogenase activity. Attempts to reactivate transhydrogenase by reduction of labelled sulfhydryls were not successful. Photo-induced transfer of the NBD adduct in partially inhibited transhydrogenase, from the sulfhydryls to reactive NH2 groups of amino-acid residue(s), identified as lysine residue(s), was parallelled by an inhibition of the residual transhydrogenase activity. It is suggested that a lysine localized close to the fast-reacting NBD-Cl-reactive sulfhydryl groups is essential for activity.

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.

Bovine heart submitochondrial particle energy-linked NADH leads to NADP+ transhydrogenase also catalyzes transhydrogenation between NADPH and NADP+. The stereochemistry of hydride ion transfer in the NADH leads to NADP+ reaction involves the nicotinamide 4A locus of NADH and the 4B locus of NADPH. In this paper it is demonstrated that during NADPH leads to NADP+ transhydrogenation the NADP+ is reduced exclusively at the 4B locus and that oxidation of NADPH is predominately at the 4B locus. Reduction of [4-3H]NADP+ by NADPH yielded [4A-3H]NADPH as the only product. Oxidation of [4A-3H]NADPH by NADP+ resulted in the conversion of nearly 33% of the label into [4-3H]NADP+, whereas the oxidation of [4B-3H] NADPH yielded only about a 6.5% conversion. These data suggest that while a small portion of total energy-linked NADPH leads to NADP+ transhydrogenation results from the binding of NADPH at the NAD domain of the active site with hydride transfer to NADP+ bound at the NADP domain, most of the reaction occurs by a mechanism in which both substrates are bound sequentially at the NADP domain. It is proposed that NADPH leads to NADP+ transhydrogenation represents a partial reaction of NADH leads to NADP+ transhydrogenation which involves the participation of a reduced-enzyme intermediate.

The pyridine nucleotide specificity, the participation of Δψ, and the energy-linked transhydrogenase in the process of Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ retention and efflux from mitochondria.

The mitochondrial electron transport system of adult Hymenolepis diminuta exhibited an apparent specificity in terms of reduced pyridine nucleotide utilization. The preferred substrate for both the minor oxidase and the physiologically required fumarate reductase system was NADH. Intramitochondrial reducing equivalents, needed for phosphorylation via the anaerobic, electron transport-dependent, fumarate reductase, were generated as NADPH by the action of the cestode's NADP-specific "malic" enzyme. However, H. diminuta mitochondria catalyzed an NADPH : NAD transhydrogenation which would serve in hydride ion transfer from NADPH to NAD, thereby producing NADH required for the anaerobic, electron transport mechanism. Accordingly, NADPH utilization was increased when NAD was added to the mitochondrial system. The most significant increase occurred in the presence of both NAD and fumarate. These data indicate a coupling of the NADPH : NAD transhydrogenase with mitochondrial electron transport. This coupling of the transhydrogenase with electron transport was demonstrated using disrupted mitochondria and mitochondrial membrane preparations. Under conditions of reduced oxygen tension, the coupling of the transhydrogenase to fumarate reduction was apparent. In adult Ascaris suum, where the "malic" enzyme physiologically utilizes NAD, the mitochondria differ from those of H. diminuta because NADPH : NAD transhydrogenase activity was minimal under the conditions of assay. The rate of NADPH utilization by the nematode mitochondrial system is not increased appreciably in the presence of NAD when either oxygen or fumarate serves as the acceptor.

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

Non-energy dependent transhydrogenase activity in submitochondrial particles is stimulated at pH 8 by some inhibitors of mitochondrial carboxylate translocases. On solubilising the enzyme or assaying it at pH 6 these inhibitors have little effect. Km values remain unchanged under all these conditions. Conversely, energy-dependent transhydrogenase activity is inhibited by these substances possibly due to their direct action on proton transport. It is suggested that the observed effects may all be due to changes in membrane conformation induced by the inhibitors.

The intramitochondrial localization of the fumarate reductase, NADPH→NAD transhydrogenase, ‘malic’ enzyme and fumarase was determined in adult Hymenolepis diminuta. The distribution of marker enzymes for the inner membrane, matrix, intermembrane space and outer membrane of H. diminuta mitochondria simulated that of the corresponding ascarid and mammalian organelles. The electron transport-coupled fumarate reductase and the NADPH→NAD transhydrogenase were components of the inner membrane whereas the ‘malic’ enzyme and fumarase were in the matrix soluble compartment. Assessments of NADH utilization, malate-dependent NADP reduction and NADPH→NAD transhydrogenation by presumedly intact and disrupted mitochondria supported the localization data. The findings presented indicate that in H. diminuta mitochondria (a) NADPH and fumarate are accumulated within the matrix compartment; (b) transhydrogenation between NADPH and NAD is an event associated with the matrix side of the inner membrane; and (c) electron transport-dependent NADH oxidation and fumarate reduction occur at sites on the matrix side of the inner membrane.

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.

Nicotinamide nucleotide transhydrogenase in rat liver submitochondrial particles is inhibited by treatment with NN'-dicyclohexylcarbodi-imide or butane-2,3-dione. Both inhibitions are pseudo-first-order with respect to enzyme activity. The reaction order with respect to inhibitor is close to unity for butanedione, but is significantly lower than unity for dicyclohexylcarbodi-imide.

The effect of pH on the kinetics of the following transhydrogenation reactions catalyzed by energized and nonenergized submitochondrial particles has been studied: NADH leads to 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP), NADH leads to thionicotinamide adenine dinucleotide phosphate (thioNADP), NADPH leads to AcPyADP, and NADPH leads to thioNADP. The effect of membrane energization on reaction rates can be approximated in the case of NADH leads to AcPyADP and thioNADP transhydrogenations, or equaled in the case of NADPH leads to AcPyADP and thioNADP transhydrogenations by lowering the assay pH to less than or equal to 6.0. For the reactions NADH leads to AcPyADP and thioNADP under energy-linked conditions, substrate Km values are lowest and Vmax values are highest at pH 7 to 7.5, the optimum pH for mitochondrial energy transduction processes. Under non-energy-linked conditions, however, Km values were lowest and Vmax values were highest at the most acid conditions (pH = 5.5) examined. Plots of ln (Vmax/Km) (an index of enzyme-substrate affinity to form a complex) versus pH showed the highest affinity at pH 7 to 7.5 for energy-linked conditions. Similar plots for non-energy-linked conditions. Similar plots for non-energy-linked conditions. Similar plots for non-energy-linked conditions showed a sharp and linear increase in the value of ln (Vmax/Km) as the assay pH was lowered from 8.5 to 6 to 6.5. This was followed by a less steep line down to pH 5.5, with a clear break at pH 6 to 6.5. These results suggested the involvement of ionizable group(s) with pK value(s) at pH 6 to 6.5 affecting enzyme-substrate binding under non-energy-linked conditions. An analogous mechanism, possibly by way of proton-induced conformation change of the transhydrogenase enzyme (EC 1.6.1.1), might be involved in increasing enzyme-substrate affinity and consequently the rate of transhydrogenation under energy-linked conditions.roton-induced conformation change of the transhydrogenase enzyme (EC 1.6.1.1), might be involved in increasing enzyme-substrate affinity and consequently the rate of transhydrogenation under energy-linked conditions.

1 The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, α and β, of apparent Mr 50000 and 47000. 2 During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine. 3 Treatment of transhydrogenase with N,N′-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N′-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [14C]Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme. 4 The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.

1 The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). 2 The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. 3 The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580–600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. 4 The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.

NADPH/NAD and NADH/NAD transhydrogenase activities have been demonstrated in the mitochondrial fraction of Taenia crassiceps cysticerci. These activities seem to result from the activities of two different enzyme systems. Both transhydrogenase activities exhibited a high heat resistence and they were completely abolished only by the temperatures higher than 100 degrees C. The activity of NADH/NAD transhydrogenase was rather high (116 nmol.min-1.mg-1 protein), but it was found to exhibit a low affinity to NADH (K'M = 1.43.10(-4) M). The physiological significance of NADH/NAD transhydrogenase is discussed.

Beef heart mitochondrial pyridine nucleotide transhydrogenase undergoes a 2-fold changes in apparent activation energy between 5 degrees and 40 degrees C, from 30 kcal/mol at the lower end to 15 kcal/mol at the upper end of this range. In order to determine whether changes in the structural domain of transhydrogenase accompany the large change in activation energy, the degree of proteolytic inactivation of membrane-bound transhydrogenase was monitored as a function of temperature. Changes in the susceptibility of transhydrogenase to proteolytic inactivation over the 5 degrees -40 degrees C interval correlate well with the observed alteration in activation energies. No temperature-dependent change in either apparent activation energy or susceptibility to proteolysis were observed in detergent-solubilized transhydrogenase. Interactions between transhydrogenase and its microenvironment in the mitochondrial membrane may be an important factor in determining its activity.

The effect of glutathione, glutathione disulfide and the dithiol reagent phenylarsine oxide on purified soluble as well as reconstituted mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. Glutathione disulfide and phenylarsine oxide caused an inhibition of transhydrogenase, the extent of which was dependent on the presence of either of the transhydrogenase substrates. In the absence of NADPH glutathione protected partially against inactivation by glutathione disulfide and phenylarsine oxide. In the presence of NADPH glutathione also inhibited transhydrogenase. Reconstituted transhydrogenase vesicles behaved differently as compared to the soluble transhydrogenase and was partially uncoupled by GSSG. It is concluded that transhydrogenase contains a dithiol that is essential for catalysis as well as for proton translocation.

Purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and NAD(H) protects the enzyme against this inhibition [Phelps, D.C., and Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The tryptic digest of TH treated with [14C]DCCD showed a single radioactive peak upon FPLC chromatography. This radioactive peak was absent from tryptic digests of TH treated with [14C]DCCD in the presence of NADH. Sequence analysis of the radioactive peak showed that it contained two peptides, one derived from the other as a result of incomplete cleavage by trypsin of a lysyl-glutamyl bond. After further digestion with Staphylococcus V8 protease, the smaller radioactive fragment was isolated and sequenced. The amino acid sequence of this fragment, as determined by manual Edman degradation, was Ala-Glu-Met-Lys. The second residue was modified. Amino acid analysis and sequence studies on the radioactive tryptic peptide mixture indicated that the sequence around the DCCD-modified residue was Glu-Met-Ser-Lys-Glu-Phe-Ile-Glu-Ala-Glu-Met-Lys. In other studies, this sequence has been found in the amino acid sequence of TH as predicted from the corresponding cDNA. The DCCD-modified peptide is near the site of NAD(H) binding, as labeled with radioactive p-fluorosulfonylbenzoyl-5'-adenosine. Furthermore, there is a high degree of homology in this region between the amino acid sequences of the bovine heart TH and the alpha subunit of the Escherichia coli TH.

Escherichia coli can normally grow aerobically in the presence of chlorate; however, mutants can be isolated that can no longer grow under these conditions. We present here the biochemical characterization of one such mutant and show that the primary genetic lesion occurs in the ubiquinone-8-biosynthetic pathway. As a consequence of this, under aerobic growth conditions the mutant is apparently unable to synthesize formate dehydrogenase, but can synthesize a Benzyl Viologen-dependent nitrate reductase activity. The nature of this activity is discussed.

Treatment of rats or liver homogenates with catecholamines (isoproterenol or noradrenaline) increased activities of both NAD+-dependent isocitrate dehydrogenase and NAD(P)+ -transhydrogenase (in the direction of hydrogen transfer NADPH → NAD+ with no change in NADP+ -dependent isocitrate dehydrogenase. These effects were realized via β-adrenoceptors. Cyclic AMP mimicked the catecholamine action on incubation with liver homogenate. The effects of catecholamines and cyclic AMP were not additive.

Butanedione in the presence of borate buffer reversibly inhibits Rhodospirillum rubrum chromatophore transhydrogenase complex and the separated membrane-bound and soluble factor components of the complex. NADP + completely protected against inactivation of the membrane-bound component, whereas NAD + was without effect. Soluble factor was maximally protected only partially by either NAD + or NADP + , but a mixture of the substrates afforded complete protection. NADP + -dependent association of soluble factor with factor-depleted membranes was markedly decreased after incubation of membranes with butanedione in the absence, but not in the presence, of NADP + . Soluble factor was bound to agarose-NAD and was eluted by NAD + , but not by NADP + . These results demonstrate the presence of at least three nicotinamide adenine dinucleotide binding sites on R. rubrum transhydrogenase complex, including separate NADP and NAD binding sites on soluble factor and a NADP binding site on the membrane-bound component.

A direct measurement of the electrogenic activity of purified mitochondrial transhydrogenase has been carried out. For this purpose beef heart transhydrogenase was isolated and reconstituted with phospholipids to form proteoliposomes. The transhydrogenase proteoliposomes were incorporated into a membrane filter impregnated with a decane solution of phospholipids. It was shown that the addition of substrates of either forward (NADPH and NAD+) or reverse (NADH and NADP+) transhydrogenase reaction gives rise to formation of electric potential difference across the proteoliposome-treated membrane filter. The electric vector depends on the direction of the reaction. The proteoliposome-supplemented compartment charges negatively in the case of the reverse one. The addition of the reaction products after substrate equalizes the potentials. The transhydrogenase-treated membrane retains the ability to the transhydrogenase-linked electrogenesis after removal of an excess of non-incorporated proteoliposomes.