Jan Rydström

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties

Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.

UV‐B‐ and oxidative stress‐induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP‐ribose)polymerase inhibitor in plant tissue

Nicotinamide and trigonelline contents increased in Catharanthus roseus tissue culture after exposure to 2,2′azobis(2‐amidinopropane) dihydrochloride (AAPH) or vanadylsulfate and in Pisum sativum leaves after exposure to UV‐B radiation. Vanadylsulfate increased phenylalanine ammonia‐lyase (PAL) activity and the content of reduced and oxidized glutathione in C. roseus tissue culture. The increases in PAL activity caused by 2 mM AAPH or 0.2 mM vanadylsulfate were prevented by 0.1 mM 3‐aminobenzamide (3‐AB), an inhibitor of poly(ADP‐ribose)polymerase. Present results support the hypothesis [Berglund T., FEBS Lett. (1994) 351, 145–149] that nicotinamide and/or its metabolites may function as signal transmittors in the response to oxidative stress in plants and that poly(ADP‐ribose)polymerase has a function in the induction of defensive metabolism.

Proton-translocating transhydrogenase: an update of unsolved and controversial issues

Proton-translocating transhydrogenases, reducing NADP+ by NADH through hydride transfer, are membrane proteins utilizing the electrochemical proton gradient for NADPH generation. The enzymes have important physiological roles in the maintenance of e.g. reduced glutathione, relevant for essentially all cell types. Following X-ray crystallography and structural resolution of the soluble substrate-binding domains, mechanistic aspects of the hydride transfer are beginning to be resolved. However, the structure of the intact enzyme is unknown. Key questions regarding the coupling mechanism, i.e., the mechanism of proton translocation, are addressed using the separately expressed substrate-binding domains. Important aspects are therefore which functions and properties of mainly the soluble NADP(H)-binding domain, but also the NAD(H)-binding domain, are relevant for proton translocation, how the soluble domains communicate with the membrane domain, and the mechanism of proton translocation through the membrane domain.

THE MEMBRANE TOPOLOGY OF PROTON-PUMPING ESCHERICHIA COLI TRANSHYDROGENASE DETERMINED BY CYSTEINE LABELING

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 α-subunit and nine in the β-subunit), with the C terminus of the α-subunit and the N terminus of the β-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. colitranshydrogenases.

Metabolism of polycyclic aromatic hydrocarbons in the rat ovary comparison with metabolism in adrenal and liver tissues

Abstract The activities of polycyclic aromatic hydrocarbon metabolizing enzymes and their regulation in the rat ovary were investigated and compared to those in the rat adrenal and liver. 7,12-Dimethylbenz[a]anthracene and benz[a]pyrene, both potent carcinogens, were converted by ovarian microsomes to hydrophilic products by the same cytochrome P-450 dependent monooxygenase with an apparent Km of about 0.05–0.1 μM. These data indicate that the ovarian AHH has a 20- and 200-fold higher affinity for polycyclic aromatic hydrocarbons than the adrenal and liver, respectively. 3-Methylcholanthrene (MC) and 7,12-dimethylbenz[a]anthracene apparently induce ovarian AHH but in an irreproducible manner, suggesting a possible regulation by endogenous factors. Among various inhibitors tested, ellipticine, SU-9055 and α-napthoflavone were the most efficient; steroids, e.g. cholesterol and estradiol, were less efficient inhibitors. A comparison of the adrenal and liver with respect to metabolite patterns for DMBA and BP reveals that there are striking qualitative as well as quantitative similarities between the adrenal and ovarian systems, whereas the liver appears to be different. Also, glutathione-S-transferase activity is inducible by MC in the liver but not in the adrenal or ovary. DT-diaphorase is induced by MC in all three organs but only by DMBA in the liver and ovary. In contrast, epoxide hydrolase is not induced by MC or DMBA in either of these organs. These results suggest that the ovarian and adrenal systems involved in bioactivation and detoxification of xenobiotics are closely related whereas those of the liver are different.

Purification and molecular properties of reconstitutively active nicotinamide nucleotide transhydrogenase from beef heart mitochondria

Abstract Nicotinamide nucleotide transhydrogenase from beef heart mitochondria was purified to homogeneity and characterized. The enzyme is devoid of other respiratory chain activities as well as flavin. Reduction of NAD + by NADPH catalyzed by reconstituted transhydrogenase generates an uncoupler-sensitive uptake of lipophilic anions, whereas the rate of reduction of NAD + by NADPH is enhanced about 13 fold by uncouplers. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate reveales that the protein consists of a single polypeptide of a molecular weight of 97,000.

Diminished NADPH transhydrogenase activity and mitochondrial redox regulation in human failing myocardium

Although the functional role of nicotinamide nucleotide transhydrogenase (Nnt) remains to be fully elucidated, there is strong evidence that Nnt plays a critical part in mitochondrial metabolism by maintaining a high NADPH-dependent GSH/GSSG ratio, and thus the control of cellular oxidative stress. Using real-time PCR, spectrophotometric and western blotting techniques, we sought to determine the presence, abundance and activity level of Nnt in human heart tissues and to discern whether these are altered in chronic severe heart failure. Left ventricular levels of the NNT gene and protein expression did not differ significantly between the non-failing donor (NF) and heart failure (HF) group. Notably, compared to NF, Nnt activity rates in the HF group were 18% lower, which coincided with significantly higher levels of oxidized glutathione, lower glutathione reductase activity, lower NADPH and a lower GSH/GSSG ratio. In the failing human heart a partial loss of Nnt activity adversely impacts NADPH-dependent enzymes and the capacity to maintain membrane potential, thus contributing to a decline in bioenergetic capacity, redox regulation and antioxidant defense, exacerbating oxidative damage to cellular proteins.

Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons

The toxic effects of polycyclic aromatic hydrocarbons (PAH) on spermatogenic cells undergoing meiotic division were investigated in vitro. Toxicity was assayed as alterations in cell nucleus morphology and cell survival and by DNA flow cytometry. Benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) inhibited the progression of spermatocytes through meiotic division and were highly cytotoxic at concentrations higher than 1 microM. These results were obtained upon addition of a drug-metabolizing system, indicating that the seminiferous tubules lack the enzymes required for the initiation of PAH metabolism. The spindle poisons, e.g., vincristine and Colcemid, a group of direct-acting agents, affected spermatogenesis during meiotic division in a manner similar to that observed with PAH. In contrast, adriamycin did not inhibit meiotic division, although it did induce the formation of meiotic micronuclei as a result of chromosome breakage. It is concluded that low concentrations, i.e., 0.1 microM of PAH, strongly inhibit meiotic division, presumably after metabolic activation to reactive molecules functionally resembling direct-acting alkylating agents. High concentrations of PAH are cytotoxic.

Expression of proton-pumping nicotinamide nucleotide transhydrogenase in mouse, human brain and C. elegans

Proton-translocating nicotinamide nucleotide transhydrogenase is located in the mitochondrial inner membrane and catalyzes the reduction of NADP(+) by NADH to NADPH and NAD(+). The present investigation describes the expression of the transhydrogenase gene in various mouse organs, subsections of the human brain and Caenorhabditis elegans. In the mouse, the expression was highest in heart tissue (100%) followed by kidney (64%), testis (52%), adrenal gland (41%), liver (35%), pancreas (34%), bladder (26%), lung (25%), ovary (21%) and brain (14%). The expression in brain tissue was further investigated in the human brain which showed a distribution that apparently varied as a function of neuronal density, a result that was supported by estimations of expression in C. elegans using Green Fluorescent Protein (GFP) controlled by the transhydrogenase promoter. GFP-expressing C. elegans lines showed a clear concentration of fluorescence to the gut, the pharyngeal-intestinal valve and certain neurons. It is concluded that the transhydrogenase gene is expressed to various extents in all cell types in mouse, human and C. elegans.

Evidence for a free-radical-dependent metabolism of 7,12-dimethylbenz(a)anthracene in rat testis

Polycyclic aromatic hydrocarbons, e.g., 7,12-dimethylbenz(a)anthracene (DMBA), cause various toxic effects in rat testis. To clarify the mechanism of action of DMBA in adult rat testis microsomes and mitochondria from this organ were investigated in vitro with respect to their capacity to metabolize DMBA. Qualitatively, both preparations showed DMBA-hydroxylase activities which were influenced by cytochrome P-450 inhibitors, chelators, and free-radical scavengers, suggesting that the DMBA metabolism was accounted for by different metabolic pathways in these organelles. Metabolism of DMBA was also accompanied by a pronounced covalent binding to both microsomal and mitochondrial protein, catalyzed primarily by a free-radical mechanism involving free or loosely bound iron which may involve superoxide anion shown to be generated by testis mitochondria. With microsomes covalent binding was markedly enhanced by added horseradish peroxidase but not by hydrogen peroxide whereas the mitochondrial binding was affected neither by added horseradish peroxidase nor by hydrogen peroxide. Antibodies raised against cytochrome P-450 c from rat liver inhibited the microsomal DMBA-hydroxylase but not the mitochondrial DMBA metabolism. It is concluded that the microsomal DMBA conversion and covalent binding are due to a mixture of cytochrome P-450 and free-radical-dependent metabolic pathways whereas the corresponding mitochondrial reaction is due mainly to a free-radical-dependent pathway. However, the data do not allow for a conclusion as to the quantitative importance of these pathways. It is proposed that both pathways may be important in DMBA-dependent testis toxicity but also in polycyclic aromatic hydrocarbon-dependent testis toxicity in general.