Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the Kd, the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Qi center, 2-n-nonyl-4-hydroxyquinoline N-oxide and for the Qo center (E)-beta-methoxyacrylate-stilbene (MOA-stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non-fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Qi and Qo center inhibitors under different redox states of the enzyme using the same experimental set up. We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Qo center, one located in the Qi center. Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Qi center is not redox-dependent. In contrast, the binding of MOA-stilbene to the Qo center is decreased after reduction of the iron-sulfur center and cytochrome c1, whereas this redox change increases the affinity for a Qo center inhibitor of the hydroxynaphthoquinone type, 3-n-undecyl-2-hydroxynaphthoquinone. From these results, aware of the fact that the inhibitory mechanism at the Qo center is a non-competitive one, we made the hypothesis of a 'catalytic switch' to explain both the bifurcation of electron flow and the inhibition at the Qo center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed 'switch' gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.
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PMID:Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration. Evidence for a 'catalytic switch' at the Qo center. 199 66

A lambda gt10 bovine brain and a lambda gt11 bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequences directly determined from the isolated 51-kDa subunit of the bovine respiratory-chain NADH dehydrogenase. Clones were isolated that encode a protein of 464 amino acids containing all the 11 partial tryptic peptide sequences determined from the 51-kDa subunit. The size and amino acid composition of this protein agree with those determined for the purified 51-kDa subunit. Furthermore, this protein contains a putative NADH-binding domain, a possible FMN-binding site, and a putative binding site for an iron-sulfur cluster. The above evidence indicates that the cloned protein is the 51-kDa subunit or its precursor. A search for sequence similarity with proteins in the Protein Identification Resource data base has revealed that the 51-kDa subunit has 32% amino acid sequence identity with a major portion of the alpha subunit of the soluble NAD(+)-reducing hydrogenase from Alcaligenes eutrophus. In particular, there are three segments of high sequence similarity (70-88%) between the two proteins which correspond to the three ligand-binding sites.
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PMID:cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase. 203 66

A systematic study of the effects of the synthetic glucocorticoid, methylprednisolone (MP), on respiration and energy coupling in tightly-coupled mitochondria isolated from rat tissues has been initiated. In intact rat skeletal muscle, liver and heart mitochondria, incubation, in vitro, with greater than or equal to 0.1 mM MP caused inhibition of the state 3 respiratory rates with succinate and NAD-linked substrates. In skeletal muscle and heart mitochondria, the oxidation of succinate was significantly more sensitive to MP than was that of the NAD-linked substrates. No effects were seen at low concentrations (less than 0.02 mM) of MP. In all three tissues, these data together with analysis of the partial reactions of the electron transport chain and steady-state kinetic analysis of cytochrome reduction indicated that in isolated mitochondria high concentrations of MP: (a) inhibit the oxidation of NAD-linked substrates at the level of the respiratory chain between the primary NADH dehydrogenase flavoprotein and coenzyme Q, most likely at the iron-sulfur centers or coenzyme Q-binding proteins of complex I; and (b) inhibit succinate oxidation in intact (but not disrupted) mitochondria, not by inhibiting electron transfer along the respiratory chain, but possibly at the level of succinate transport into the mitochondria. The results of these studies suggest that the therapeutic effects of MP in mitochondrial disease result from indirect effects rather than direct effects on the mitochondrial membrane. More importantly, the absence of an effect at low MP concentrations provides the baseline information needed for further studies to be carried out in vivo.
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PMID:In vitro effects of glucocorticoid on mitochondrial energy metabolism. 204 73

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.
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PMID:Studies on the hyperplasia ('regeneration') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects. 210 18

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. When the Paracoccus NADH dehydrogenase complex was irradiated by UV light in the presence of [adenylate-32P]NAD, radioactivity was incorporated exclusively into one of three polypeptides of Mr approximately 50,000. Similar results were obtained when [adenylate-32P]NADH was used. The labeling of the Mr 50,000 polypeptide was diminished when UV irradiation of the enzyme with [adenylate-32P]NAD was performed in the presence of NADH, but not in the presence of NADP(H). The labeled polypeptide was isolated by preparative sodium dodecyl sulfate gel electrophoresis and was shown to cross-react with antiserum to the NADH-binding subunit (Mr = 51,000) of bovine NADH-ubiquinone oxidoreductase. Its amino acid composition was also very similar to that of the bovine NADH-binding subunit. These chemical and immunological results indicate that the Mr 50,000 polypeptide is an NADH-binding subunit of the Paracoccus NADH dehydrogenase complex.
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PMID:Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans. 211 69

The respiratory chain NADH:ubiquinone oxidoreductase (NADH dehydrogenase or Complex I) of mitochondria comprises some 30 different subunits, and one FMN and 4 or 5 iron-sulfur clusters as internal redox groups. The bacterial glucose dehydrogenase, which oxidizes glucose to gluconolactone in the periplasmatic space and transfers the electrons to ubiquinone, is a single polypeptide chain with pyrolloquinoline quinone as the only redox group. We report here that the two different enzymes have the same ubiquinone binding domain motif and we discuss the predicted membrane folding of this domain with regard to its role in the proton translocating function of the two enzymes.
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PMID:The same domain motif for ubiquinone reduction in mitochondrial or chloroplast NADH dehydrogenase and bacterial glucose dehydrogenase. 214 3

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.
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PMID:Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8. 215 29

ICRF-187 ((+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane) has shown promise (Speyer et al., N. Engl. J. Med. 319, 745 (1988] as a cardioprotective agent against what may be an iron-based adriamycin-induced cardiotoxicity. ICRF-187, which is membrane permeable, likely exerts its action through its rings-opened hydrolysis product which has a structure similar to EDTA and which, likewise, strongly binds metal ions. Both Fe3(+)-adriamycin and Cu2(+)-adriamycin reacted directly with ICRF-187, promoting a ring-opening hydrolysis of ICRF-187 that resulted in the displacement of the metal ion from its complex with adriamycin. Thus ICRF-187 can be considered to be acting as a "suicide protective agent" in its reaction with metal ion-adriamycin complexes. That this metal ion complex-promoted hydrolysis was preceded by mixed ligand complex formation is evidenced by the fact that the first-order rate constant for loss of metal ion from the adriamycin complex exhibits saturation behaviour at high ICRF-187 concentrations. Also direct spectroscopic evidence was obtained both for a Cu2(+)-adriamycin-ICRF-187 mixed ligand complex and a Cu2+ (ICRF-187)2 complex. The Fe3(+)-adriamycin complex inactivates the cytochrome c oxidase and NADH cytochrome c reductase activity on submitochondrial particles. The protection that ICRF-187 affords against this loss of activity may be explained both on the basis of simple Fe3+ removal from Fe3(+)-adriamycin and also on formation of a less active Fe3(+)-adriamycin-ICRF-187 mixed ligand complex.
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PMID:The iron(III) and copper(II) complexes of adriamycin promote the hydrolysis of the cardioprotective agent ICRF-187 ((+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane). 216 Jan 91

The pre-steady-state redox reactions of the Rieske iron-sulfur protein isolated from beef heart mitochondria have been characterized. The rates of oxidation by c-type cytochromes is much faster than the rate of reduction by ubiquinols. This enables the monitoring of the oxidation of ubiquinols by the Rieske protein through the steady-state electron transfer to cytochrome c in solution. The pH and ionic strength dependence of this reaction indicate that the ubiquinol anion is the direct reductant of the oxidized cluster of the iron-sulfur protein. The second electron from ubiquinol is diverted to oxygen by the isolated Rieske protein, and forms oxygen radicals that contribute to the steady-state reduction of cytochrome c. Under anaerobic conditions, however, the reduction of cytochrome c catalyzed by the protein becomes mechanicistically identical to the chemical reduction by ubiquinols. The present kinetic work outlines that: (i) the electron transfer between the ubiquinol anion and the Rieske cluster has a comparable rate when the protein is isolated or inserted into the parent cytochrome c reductase enzyme; (ii) the Rieske protein may be a relevant generator of oxygen radicals during mitochondrial respiration.
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PMID:The oxidation of ubiquinol by the isolated Rieske iron-sulfur protein in solution. 217 22

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.
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PMID:Iron-reductases in the yeast Saccharomyces cerevisiae. 218 97


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