NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta.

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.

Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli.

Pyridine nucleotide transhydrogenases of bacterial cytosolic membranes and mitochondrial inner membranes are proton pumps in which hydride transfer between NADP(+) and NAD(+) is coupled to proton translocation across cytosolic or mitochondrial membranes. The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two subunits (alpha and beta). Three domains are recognized. The extrinsic cytosolic domain 1 of the amino-terminal region of the alpha subunit bears the NAD(H)-binding site. The NADP(H)-binding site is present in domain 3, the extrinsic cytosolic carboxyl-terminal region of the beta subunit. Domain 2 is composed of the membrane-intrinsic carboxyl-terminal region of the alpha subunit and the membrane-intrinsic amino-terminal region of the beta subunit. Treatment of the transhydrogenase of E. coli with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) inhibited enzyme activity. Analysis of inhibition revealed that several sites on the enzyme were involved. NBD chloride modified two (betaCys-147 and betaCys-260) of the seven cysteine residues present in the transhydrogenase. Modification of betaCys-260 in domain 2 resulted in inhibition of enzyme activity. Modification of residues other than cysteine residues also resulted in inhibition of transhydrogenation as shown by use of a cysteine-free mutant enzyme. The beta subunit was modified by NBD chloride to a greater extent than the alpha subunit. Reaction of domain 2 and domain 3 was prevented by NADPH. Modification of domain 3 is probably not associated with inhibition of enzyme activity. Modification of domain 2 of the beta subunit resulted in a decreased binding affinity for NADPH at its binding site in domain 3. The product resulting from the reaction of NBD chloride with NADPH was a very effective inhibitor of transhydrogenation. In experiments with NBD chloride in the presence of NADPH it is likely that all of the sites of reaction described above will contribute to the inhibition observed. The NBD-NADPH adduct will likely be more useful than NBD chloride in investigations of the pyridine nucleotide transhydrogenase.

Inhibition of proton-translocating transhydrogenase from photosynthetic bacteria by N,N'-dicyclohexylcarbodiimide.

The effects of N,N'-dicyclohexylcarbodiimide [(cHxN)2C] on the proton-translocating enzyme, NAD(P) H(+)-transhydrogenase (H(+)-Thase), from two species of phototrophic bacteria have been investigated. The polypeptides of H(+)-Thase from Rhodobacter capsulatus are membrane-associated, requiring detergent to maintain solubility. The enzyme from Rhodospirillum rubrum, however, has a water soluble polypeptide (Ths) and a membrane-associated component (Thm) which, separately, have no activity but which can be fully reconstituted to give a functional complex. Two observations suggest that (cHxN)2C inhibited H(+)-Thase from both species by modification either close to or at the NADP(H)-binding site on the enzyme: (a) the presence of NADP+ or NADPH caused increased inhibition by (cHxN)2C and (b) after treatment of the purified enzyme from Rb. capsulatus with (cHxN)2C, the release of NADP+ became rate-limiting, as evidenced by a stimulated rate of NADPH-dependent reduction of acetylpyridine adenine dinucleotide by NADH. Experiments in which Ths and Thm from R. rubrum were separately treated with (cHxN)2C then reconstituted with the complementary, untreated component revealed that the NADP(H)-enhanced modification by (cHxN)2C was confined to Thm. In contrast to some experiments with mitochondrial H(+)-Thase [Wakabayashi, S. & Hatefi, Y. (1987) Biochem. Int. 15, 667-675], there was no protective effect of either NAD+ or NADH on the inhibition by (cHxN)2C of enzyme from photosynthetic bacteria. However, amino acid sequence analysis of proteolytic fragments of Ths revealed that the NAD(H)-protectable, (cHxN)2C-reactive glutamate residue in mitochondrial H(+)-Thase might be replaced by glutamine in R. rubrum.

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., F1-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 to 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.

[The role of inner membrane in the realization of cAMP-dependent activation of mitochondrial enzymes]. / Znachenie vnutrennei membrany v realizatsii cAMP-zavisimoi aktivatsiifermentov mitokhondrii.

It was shown that the increase in the activities of transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase after incubation of mitochondria with cAMP is due to the stimulating effect of cAMP on mitochondria, but not to the increased stability of mitochondria to the incubation procedure. Treatment of mitochondria with trypsin prevents the action of cAMP on the both enzymes. The integrity of the inner mitochondrial membrane is necessary for the manifestation of cAMP effect. Pretreatment of mitochondria with the local anesthetic, lidocaine, prevents the activation of NAD(P)(+)-transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase during subsequent incubation of mitochondria with cAMP. It is concluded that the role of the inner mitochondrial membrane consists in the reception of the cAMP signal for the internal compartment of mitochondria, i.e. for mitoplasts. Peripheral protein(s) on the external side of the inner mitochondrial membrane seems to play a role in cAMP reception.

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893.

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational features of bovine heart mitochondrial transhydrogenase.

Both purified and functionally reconstituted bovine heart mitochondrial transhydrogenase were treated with various sulfhydryl modification reagents in the presence of substrates. In all cases, NAD+ and NADH had no effect on the rate of inactivation. NADP+ protected transhydrogenase from inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in both systems, while NADPH slightly protected the reconstituted enzyme but stimulated inactivation in the purified enzyme. The rate of N-ethylmaleimide (NEM) inactivation was enhanced by NADPH in both systems. The copper-(o-phenanthroline)2 complex [Cu(OP)2] inhibited the purified enzyme, and this inhibition was substantially prevented by NADP+. Transhydrogenase was shown to undergo conformational changes upon binding of NADP+ or NADPH. Sulfhydryl quantitation with DTNB indicated the presence of two sulfhydryl groups exposed to the external medium in the native conformation of the soluble purified enzyme or after reconstitution into phosphatidylcholine liposomes. In the presence of NADP+, one sulfhydryl group was quantitated in the nondenatured soluble enzyme, while none was found in the reconstituted enzyme, suggesting that the reactive sulfhydryl groups were less accessible in the NADP+-enzyme complex. In the presence of NADPH, however, four sulfhydryl groups were found to be exposed to DTNB in both the soluble and reconstituted enzymes. NEM selectively reacted with only one sulfhydryl group of the purified enzyme in the absence of substrates, but the presence of NADPH stimulated the NEM-dependent inactivation of the enzyme and resulted in the modification of three additional sulfhydryl groups. The sulfhydryl group not modified by NEM in the absence of substrates is not sterically hindered in the native enzyme as it can still be quantitated by DTNB or modified by iodoacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)

NBD-Cl modification of essential residues in mitochondrial nicotinamide nucleotide transhydrogenase from bovine heart.

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.

ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles.

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.

Evidence for a role of a vicinal dithiol in catalysis and proton pumping in mitochondrial nicotinamide nucleotide transhydrogenase.

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.

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 0.6 mM). Xylitol partially reversed this inhibitory effect. The inhibition was not mediated by a direct effect of oleate on microsomal components since concentrations of oleate ranging up to 1.0 mM did not affect p-nitroanisole O-demethylation by isolated microsomes. 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 CoA inhibited p-nitroanisole O-demethylation in microsomes (Ki about 30 microM), and both oleoyl CoA and palmitoyl CoA inhibited the energy-linked nicotinamide nucleotide transhydrogenase in submitochondrial particles (Ki about 1 microM). 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.

The effects of some mitochondrial translocase inhibitors on the activity of rat liver transhydrogenase.

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.

Inhibition of the mitochondrial nicotinamide nucleotide transhydrogenase by dicyclohexylcarbodiimide and diethylpyrocarbonate.

The mitochondrial nicotinamide nucleotide transhydrogenase enzyme (EC 1.6.1.1) is inhibited by treatment with dicyclohexylcarbodiimide or diethylpyrocarbonate. Both inhibitions are pseudo first order with respect to incubation time, and both reaction orders with respect to inhibitor concentration are close to unit, indicating that in each case inhibition results from the binding of one inhibitor molecule per active unit of the transhydrogenase enzyme. In the presence of either inhibitor, both the energy-linked and the nonenergy-linked transhydrogenation reactions are inhibited at about the same rate. The water-soluble carbodiimide, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide, showed no inhibition, however, NAD(H) and reduced or oxidized 3-acetylpyridine adenine dinucleotide protected the enzyme against inhibition by dicyclohexylcarbodiimide, while NADP (but not NADPH) appeared to increase the rate of inhibition. Substrates did not protect the enzyme against inhibition by diethylpyrocarbonate. [14C]dicyclohexylcarbodiimide labeled the transhydrogenase enzyme in submitochondrial particles. Treatment of labeled particles with trypsin resulted in fragmentation of the transhydrogenase enzyme and loss of a labeled polypeptide of Mr = approximately 100,000 as determined by polyacrylamide gel electrophoresis.

Steady-state kinetics and the inactivation by 2,3-butanedione of the energy-independent transhydrogenase of Escherichia coli cell membranes.

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.