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1.
We have examined the substrate specificity and inhibitor sensitivity of H2O2 formation by rat heart mitochondria. Active H2O2 production requires both a high fractional reduction of Complex I (indexed by NADH/NAD+ + NADH ratio) and a high membrane potential, . These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H2O2 formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn2+ to give 10 nmol/mg of mitochondrial protein enhances H2O2 production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H2O2 production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).  相似文献   

2.
The rotenone sensitivity of bovine heart NADH: coenzyme Q oxidoreductase (Complex I) depends significantly on coenzyme Q1 concentration. The rotenone-insensitive Complex I reaction in Q1 concentration range above 300 M indicates an ordered sequential mechanism with Q1 and reduced Q1 (Q1H2) as the initial substrate to bind to the enzyme and the last product to be released from the enzyme product complex, respectively. This is the case in the rotenone-sensitive reaction although both K m and V max values of the rotenone-insensitive reaction for Q1 are significantly higher than those of the rotenone-sensitive reaction (Nakashima et al., 2002, J. Bioenerg. Biomemb. 34, 11–19). This rigorous control mechanism between the nucleotide and ubiquinone binding sites strongly suggests that the rotenone-insensitive reaction is also physiologically relevant.  相似文献   

3.
The kinetics of the NADH3'-acetylpyridine adenine dinucleotide (APAD+) transhydrogenase reaction (DD-reaction) catalyzed by different preparations of mitochondrial NADH-dehydrogenase (submitochondrial particles (SMP), purified Complex I, and three-subunit fragment of Complex I (FP)) have been studied. Complex I (in SMP or in purified preparation) catalyzes two NADHAPAD+ reactions with different rates and nucleotide affinities. Reaction 1 has high affinity to APAD+ (K m = 7 M, for SMP) and low rate (V m = 0.2 mol/min per mg protein, for SMP) and occurs with formation of a ternary complex. Reaction 2 has much higher rate and considerably lower affinity for oxidized nucleotide (V m = 1.7 mol/min per mg protein and K m = 160 M, for SMP). FP catalyzes only reaction 1. ADP-ribose inhibits reaction 1 with mixed type inhibition (competitive with non-competitive) with respect to NADH and APAD+. Rhein competes with both substrates. The results suggest that at least two nucleotide-binding sites exist in Complex I.  相似文献   

4.
Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H2O2). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O2/H2O2 formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O2/H2O2 formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H2O2. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O2/H2O2 production by KGDHC, induced a ~ 86% and ~ 84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O2/H2O2 formation by ~ 45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O2/H2O2 production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H2O2.  相似文献   

5.
Five novel silver (Ι) complexes with 1,3-imidazolidine-2-thione ligand formulated as [Ag21-SC3H6N2)6]SO4 (1), [Ag22-SC3H6N2)21-SC3H6N2)4](NO3)2 (2), [Ag(μ2-SC3H6N2)Br]n (3), [Ag22-SC3H6N2)3(CH3COO)2]n (4), [Ag(μ2-SC3H6N2)Cl]n (5) have been synthesized under similar conditions except for different anions and solvents. They are structurally characterized by elemental analysis, IR, TG and single crystal X-ray diffraction. Complex 1 is a binuclear complex, with SO42− anions as the counteranions. Complex 2 is a molecular binuclear silver cluster, with NO3 anions as the counteranions. Complex 3 shows a novel 2D lamella structure constructed by the S atom of 1,3-imidazolidine-2-thione ligand and Br anions, and exhibits 12-membered metallacyclic rings in its structure. Complex 4 is a novel 2D layered polymer with a graphite-like array of silver (I) ions, with CH3COO anion coordinated to silver(I) ions by O atom. Complex 5 shows a 3D diamond-like network structure constructed by the sulfur atom of 1,3-imidazolidine-2-thione ligand and Cl anions.  相似文献   

6.
In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 and 75-kDa subunits was detected under the conditions of O2 production, and mass spectrometry confirmed that formation of Complex I PrS mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS (Kang et al., Free Radical Biol. Med. 52:962–973; 2012). In this investigation, we tested our hypothesis in the murine heart of eNOS−/−. The eNOS−/− mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS−/− myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in O2 generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS−/− murine heart. When Complex I from the mitochondria of the eNOS−/− murine heart was analyzed by immunospin trapping and probed with anti-GSH antibody, both PrS and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS, supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS is a unique pathway for the redox regulation of mitochondrial function in vivo.  相似文献   

7.
The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 µM) and one with lower affinity (Kd = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 µM). The binding of [35S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 µM) and six at the liver enzyme (Kd = 12 µM). The Paracoccus enzyme had only one binding site (Kd = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)  相似文献   

8.
Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K i =ca. 0.5 µM) was considerably less than that of antimycin (K i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [3H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [3H]DAA, respectively. The labeling of band 7 by [3H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [3H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.  相似文献   

9.
Two tetra-nuclear Ag(I) complexes with styrene-functionalized N-heterocyclic carbene [AgL2]2[Ag2X4] (L = 1-methyl-3-(4-vinylbenzyl)imidazol-2-ylidene, X = Cl, 2a; X = I, 2b) were prepared by the reactions between the corresponding imidazolium salts with Ag2O. The reaction mixture was further treated with AgBF4 to give a mononuclear ion-pair complex [AgL2][BF4] (3). The molecular structures of these new Ag(I) complexes have been determined by X-ray diffraction analyses. 2a and 2b consist of two [Ag(L)2]+ fragments with the central [Ag2X4]2− anion held together by the close Ag(I)-Ag(I) interactions. Complex 3 is a mononuclear ion-pair complex with a linear bi-coordinate Ag fragment.  相似文献   

10.
[Cu(bapp)ClO4]+ (1) and [Cu(bapp)Cl]+ (2) were prepared by the reaction of 1,4-bis(3-aminopropyl)piperazine (bapp) with copper acetate and copper chloride in the presence of sodium perchlorate, respectively, and structurally characterized. Complex 1 has a square-pyramidal geometry, whereas [Cu(3,2,3-tet)(ClO4)]+ (A) has a polymeric octahedral geometry in its X-ray crystal structure. Complex 1 is stable against disproportionation, whereas complex A is unstable in the mono-valent Cu(I) state. An aqua ligand on complex 1 in aqueous solution is substituted by NO with kinetic constants of kf=43±2 M−1 s−1 and kb=(8.8±0.2)×10−2 s−1 at 25 °C, whereas there were no spectral changes observed for complex A in saturated NO solution.  相似文献   

11.
The reaction in water of silver oxide with N-acetylglycine (H2acgly) possessing the partial structure, i.e. the OC-N-C-COOH moiety, afforded light-stable and water-soluble dinuclear silver(I) complex {[Ag(Hacgly)]2} (complex 1). X-ray crystallography revealed that complex 1 in the solid state formed a ladder polymeric structure based on bis(carboxylato-O,O′)-bridged centrosymmetric Ag2O4 core, which was different from the two previously reported structures of silver(I) glycinate. Complex 1 which only comprises labile Ag-O bonding showed a wide range of antimicrobial activities against selected bacteria, yeasts and molds. Complex 1 can also work as an useful silver(I) precursor for novel silver(I) cluster synthesis.  相似文献   

12.
Summary [125I]Insulin (porcine) binding to an epithelial cell line established from a Chinese hamster kidney, CHK-ACE−100, showed an optimum at pH 8.0 and reached a maximum after 2.5 h incubation at 25°C. Dissociation of bound [125I]insulin was facilitated by the addition of unlabeled insulin in the dilution buffer. Porcine insulin effectively competed for [125I]insulin binding to the cultured cells and was 30 and 90 times as potent as guinea pig insulin and porcine proinsulin in causing 50% inhibition of [125I]insulin binding; glucagon was completely ineffective. Scatchard analysis of the binding data yielded a curvilinear plot and a capacity of 0.6 ng/106 cells; the average affinity of the empty receptor, , was calculated to be 1.78×108 M −1 and that of the filled receptor, , 0.57×108 M −1. Substitution of fetal bovine serum (FBS) in the culture medium with bovine calf, bovine newborn, or bobby calf serum altered insulin binding characteristics in the cells and reduced cell growth. Insulin binding characteristics of cells grown in hormone-supplemented medium containing 0 to 0.1% FBS were similar to those of cells grown in minimum essential medium (MEM) containing 2 to 5% FBS. The data indicated that the established Chinese hamster kidney epithelial cell line CHK-ACE−100 possessed specific insulin receptors and the characteristics of the receptors could be manipulated by changing the serum in culture medium.  相似文献   

13.
The subunits of the F0 membrane sector of bovine heart mitochondrial H+-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H+-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry.  相似文献   

14.
15.
The 2:1 and 1:2 adducts of Au(I) and 1:2 adducts of Ag(I) with the diphosphine 2,3-bis(diphenylphosphino)maleic acid (dpmaa) have been prepared in high yields. Crystal structures have been determined for the neutral digold complex (AuCl)2(dpmaa) · 2thf (1) and the bis-chelated complex [Au(dpmaa)2]Cl · H2O · CH3OH (2). For 1, conformational rigidity imposed by the ethylenic bridge facilitates the formation of short intramolecular Au-Au contacts with no evidence of similar intermolecular contacts. Complex 2 crystallizes with [Au(dpmaa)2]+ cations hydrogen bonded through the carboxyl groups to a water molecule and chloride anion to form a H-bonded chain along the a axis. 31P NMR titration of 1 with dpmaa in acetone shows conversion to 2 at Au:P-P ratios less than 1:1 indicating similar high thermodynamic and kinetic stabilities to other bis-chelated [Au(P-P)2]+ complexes containing 5- or 6-membered chelate rings. The ionic Au(I) complex 2 and the analogous Ag(I) complex [Ag(dpmma)2]NO3 (3) are highly water soluble. The in vitro cytotoxic activity of 2 was assessed against eight different cell lines and no significant activity was found. The solubility properties and solution behaviour of the complexes are compared to the analogous 1,2-bis(diphenylphosphino)ethane (dppe) complexes and the potential significance of these results to the antitumour properties of chelated 1:2 Au(I)diphosphine complexes are discussed.  相似文献   

16.
The synthesis of an unsymmetrical phenanthroline-based ligand, 2-methyl-9-(3,5-dimethylpyrazolylmethyl)-1,10-phenanthroline (L), and its cupric [Cu(II)] (1) and cuprous [Cu(I)] (2) complexes, are reported. The X-ray structures of each of these Cu complexes show distinct changes in coordination environments consistent with the geometrical preferences of the two oxidation states. In the solid-state, the Cu(II) complex (1) adopts a geometry best described as trigonal bipyramidal, while the Cu(I) complex (2) consists of a single dicationic dimer in which the ligand bridges between two copper ions, separated by 4.26 Å. The two Cu(I) coordination sites differ in 2 with one copper center complexed in a trigonal planar geometry and the other copper in a distorted tetrahedral environment; the latter coordination results from an additional CH3CN ligand. Complex 1 exhibits a reversible redox process at −0.34 V versus Fc/Fc+ in CH3CN, attributable to the Cu2+/Cu+ couple, while the dimeric Cu(I) complex (2) does not display this redox couple on the CV timescale. Over minutes however, complex 1 does oxidize in the presence of dioxygen to 2 in CH3CN.  相似文献   

17.
Direct reaction of copper(I) chloride with triphenylphosphine (tpp) in molar ratio 2:3 and 1:3, results in the formation of the [(tpp)Cu(μ2-Cl)2Cu(tpp)2] (1) and {[CuCl(tpp)3]·(CH3CN)} (2) complexes. The complexes have been characterized by melting point, FT-IR, UV-Vis spectroscopic data and X-ray crystallography. Complex 1 is di-nuclear. Two μ2-Cl atoms bridge two copper(I) ions with tetrahedral and trigonal geometry respectively. The short copper-copper bond distance of 2.9039(6) ? in case of 1 indicates d10-d10 interaction between metal centers. Thus, our studies were extended here in the determination of the quasi-aromaticity, which results in strong Cu-Cu interactions, using the computational method of nucleus-independent chemical shifts (NICS). The NICS calculated at the inner region of the Cu2Cl2P3 core in complex 1 is shielded up to −6.05 ppm. Complex 2 is mono-nuclear where three phosphorus and one chloride atoms form a tetrahedron around the copper(I) ion. Photolysis of both complexes 1 and 2, results in the formation of triphenylphosphine oxide.The complexes 1 and 2, were tested for their in vitro cytotoxic activity against leiomyosarcoma cells (LMS) and human breast adenocarcinoma cells (MCF-7). The type of LMS cell death caused by the complexes was also evaluated by use of a flow cytometry assay. The results show that at concentration of 5 μΜ of complexes 1 and 2, 34.1% (1) and 19.6 (2)% of LMS cells undergo programmed cell death (apoptosis), while at 10 μΜ, 80.4% (1) and 65.2% (2) of LMS cells undergo apoptosis. The light sensitivity of the complex is discussed in relation with the biological activity.  相似文献   

18.
N,N-bis[4-(2-aminoethyl)morpholino]glyoxime (H2L) (Fig. 1), has been prepared in various yields using three different methods. The most efficient of these methods is the technique of microwave irradiation. The crystal structures of H2L, and of two nickel(II) complexes 1 and 2 have been determined by single crystal X-ray diffraction. Both nickel(II) complexes have a metal-ligand ratio of 1:2 in which the ligand coordinates through the two nitrogen atoms as do most vic-dioximes. The nickel(II) complexes are either hydrogen (1) or boron diphenyl bridged (2). Complex 1 was synthesized by reacting H2L with nickel(II) chloride in refluxing ethanol. Complex 2 was prepared at room temperature in an ethanol solution containing excess NaBPh4. Elemental analyses, NMR(1H, 13C), IR and mass data are also presented.  相似文献   

19.
The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676–1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the individual iron-sulfur clusters in the subunits. The preceding paper described a working model based on the kinetics with NADPH. This model is at variance with current views in the field. The present paper provides a critical overview on the possible causes for the discrepancies. It is concluded that the stability of all purified preparations described thus far, including Hatefi’s Complex I, is compromised due to removal of the enzyme from the protective membrane environment. In addition, most preparations described during the last two decades are purified by methods involving synthetic detergents and column chromatography. This results in delipidation, loss of endogenous quinones and loss of reactions with (artificial) quinones in a rotenone-sensitive way. The Fe:FMN ratio’s indicate that FMN-a is absent, but that all Fe-S clusters may be present. In contrast to the situation in bovine SMP and Hatefi’s Complex I, three of the six expected [4Fe-4S] clusters are not detected in EPR spectra. Qualitatively, the overall EPR lineshape of the remaining three cubane signals may seem similar to that of Hatefi’s Complex I, but quantitatively it is not. It is further proposed that point mutations in any of the TYKY, PSST, 49-kDa or 30-kDa subunits, considered to make up the delicate structural heart of Complex I, may have unpredictable effects on any of the other subunits of this quartet. The fact that most point mutations led to inactive enzymes makes a correct interpretation of such mutations even more ambiguous. In none of the Complex-I-containing membrane preparations from non-bovine origin, the pH dependencies of the NAD(P)H→O2 reactions and the pH-dependent reduction kinetics of the Fe-S clusters with NADPH have been determined. This excludes a proper discussion on the absence or presence of FMN-a in native Complex I from other organisms.  相似文献   

20.
Mammalian NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial inner membrane catalyzes the oxidation of NADH in the matrix. Excess NADH reduces nine of the ten prosthetic groups of the enzyme in bovine-heart submitochondrial particles with a rate of at least 3,300 s−1. This results in an overall NADH→O2 rate of ca. 150 s−1. It has long been known that the bovine enzyme also has a specific reaction site for NADPH. At neutral pH excess NADPH reduces only three to four of the prosthetic groups in Complex I with a rate of 40 s−1 at 22 °C. The reducing equivalents remain essentially locked in the enzyme because the overall NADPH→O2 rate (1.4 s−1) is negligible. The physiological significance of the reaction with NADPH is still unclear. A number of recent developments has revived our thinking about this enigma. We hypothesize that Complex I and the Δp-driven nicotinamide nucleotide transhydrogenase (Nnt) co-operate in an energy-dependent attenuation of the hydrogen-peroxide generation by Complex I. This co-operation is thought to be mediated by the NADPH/NADP+ ratio in the vicinity of the NADPH site of Complex I. It is proposed that the specific H2O2 production by Complex I, and the attenuation of it, is of importance for apoptosis, autophagy and the survival mechanism of a number of cancers. Verification of this hypothesis may contribute to a better understanding of the regulation of these processes.  相似文献   

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