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The immunoaffinity chromatography of total membrane proteins fromEscherichia colihelped determine the specificity of the monoclonal antibody 3A6 that was obtained upon immunization of mice with nicotinamide nucleotide transhydrogenase preparations and reacted with an unknownE. coliantigen. Proteins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified byN-terminal sequencing as the subunits of nitrate reductase. This conclusion was confirmed by immunoblotting with the 3A6 antibody of the proteins from theE. colicells grown upon induction of nitrate reductase. It was shown that the 3A6 antibody specifically recognizes the α subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity.

The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the alpha-subunit and nine in the beta-subunit), with the C terminus of the alpha-subunit and the N terminus of the beta-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H). A model defining betaA, alphaB, betaB, betaD, and betaE of this domain is presented. To test this model, four single cysteine mutants (cfbetaA348C, cfbetaA390C, cfbetaK424C, and cfbetaR425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, beta-Ala348, beta-Arg350, beta-Ala390, beta-Asp392, and beta-Lys424 are located in or close to the NADP(H) site. More specifically, beta-Ala348 succeeds betaB. The remarkable reactivity of betaR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). beta-Ala390 and beta-Asp392 terminate or succeed betaD, and are thus, together with the region following betaA, creating the switch point crevice where NADP(H) binds. beta-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase.

The cardiotoxic effect of daunorubicin was investigated using cultured heart cells from neonatal rat hearts. Exposure of the cells to low but toxic concentrations of daunorubicin led to drastically altered permeability properties of the plasma membrane as measured by release of lactate dehydrogenase. Simultaneously, 99Tcm-gluconate, an isotope complex used in scintigraphic imaging of myocar-dial infacts, was accumulated in the cells. The results suggest that daunorubicin-induced cardiac damage may be estimated by isotope uptake.

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.

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.

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.

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.

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.

99mTc-gluconate has previously been shown to bind to isolated rat heart mitochondria. In the presence of potassium cyanide this binding is enhanced several fold, an effect which has been shown to be correlated to the efficiency of the in vivo binding of the isotope complex in ischemic dog hearts. The present investigation shows that the potassium cyanide-induced binding was mainly localized to the mitochondrial inner membrane; in the presence of cyanide other organelles, e.g., nuclei and liver microsomes also showed some binding. Boiling, lipid extraction, or addition of N-ethylmaleimide caused a variable inhibition of the binding of 99mTc-gluconate to rat heart mitochondria, which also was markedly influenced by temperature, pH, time, and concentrations of protein and isotope complex, but not by conditions affecting energy levels or calcium transport. Fractionation of submitochondrial particles exposed to 99mTc-gluconate in the presence of potassium cyanide indicated that the 99mTc-gluconate-binding component cofractionated with cytochrome oxidase. It is postulated that a protein component localized in the mitochondrial inner membrane, possibly cytochrome oxidase, is responsible for the binding of 99mTc-gluconate.