Biomedical subjects

N Capitanio

Publications and source records attributed to N Capitanio.

35 records · Page 2Linked to original sources

PubMed1994

Mechanistic and phenomenological features of proton pumps in the respiratory chain of mitochondria.

Various direct, indirect (kinetic and thermodynamic), and combined mechanisms have been proposed to explain the conversion of redox energy into a transmembrane protonmotive force (delta p) by enzymatic complexes of respiratory chains. The conceptual evolution of these models is examined. The characteristics of thermodynamic coupling between redox transitions of electron carriers and scalar proton transfer in cytochrome c oxidase and its possible involvement in proton pumping is discussed. Other aspects dealt with in this paper are: (i) variability of <--H+/e- stoichiometries, in cytochrome c oxidase and cytochrome c reductase and its mechanistic implications; (ii) possible models by which the reduction of dioxygen to water at the binuclear heme-copper center of protonmotive oxidases can be directly involved in proton pumping. Finally a unifying concept for proton pumping by the redox complexes of respiratory chain is presented.

Animals↗

PubMed1992

pH changes associated with cytochrome c oxidase reaction with H2O2. Protonation state of the peroxy and oxoferryl intermediates.

pH changes associated with the mitochondrial cytochrome oxidase reaction with H2O2 have been studied. In the presence of ferricyanide or Tris-phenanthroline complex of CoIII as electron acceptors, reaction of H2O2 with the oxidized cytochrome oxidase is accompanied by a steady proton release with a rate constant of ca. 3 M-1.s-1 at pH 6.8. The acidification is completely inhibited by superoxide dismutase and its pre-steady-state kinetics correlates with that of the oxoferryl compound (F) accumulation. Apparently, the proton release is linked to superoxide generation by cytochrome oxidase under these conditions. In the presence of superoxide dismutase and without the electron acceptors, the H2O2-induced transitions of cytochrome oxidase from the oxidized to the peroxy (P) and from the peroxy to the oxoferryl state are not associated with any significant proton release or uptake. The results point to the following mechanism of O2- generation and protonation states of the cytochrome oxidase compounds P and F: [formula: see text]

Animals↗

PubMed1991

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

Animals↗

PubMed1991

The cytochrome chain of mitochondria exhibits variable H+/e- stoichiometry.

A study is presented of the ----H+/e- stoichiometry for H+ pumping by the cytochrome chain in isolated rat liver mitochondria under level-flow and steady-state conditions. It is shown that the ----H+/e- stoichiometry for the cytochrome chain varies under the influence of the flow rate and transmembrane delta microH+. The rate-dependence is shown to be associated with cytochrome c oxidase, whose ----H+/e- ratio varies from 0 to 1, whilst the ----H+/e- ratio for the span covered by cytochrome c reductase is invariably 2.

Animals↗

PubMed1990

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme.

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.

Animals↗

PubMed1990

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes.

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.

Animals↗

PubMed1990

Cytochrome oxidase assembly in yeast requires the product of COX11, a homolog of the P. denitrificans protein encoded by ORF3.

The synthesis of cytochrome oxidase in Saccharomyces cerevisiae was recently shown to require a protein encoded by the nuclear gene COX10. This protein was found to be homologous to the putative protein product of the open reading frame ORF1 reported in one of the cytochrome oxidase operons of Paracoccus denitrificans. In the present study we demonstrate the existence in yeast of a second nuclear gene, COX11, whose encoded protein is homologous to another open reading frame (ORF3) present in the same operon of P. denitrificans. Mutations in COX11 elicit a deficiency in cytochrome oxidase. In this and in other respects cox11 and cox10 mutants have very similar phenotypes. An antibody has been obtained against the yeast COX11 protein. The antibody recognizes a 28 kd protein in yeast mitochondria, consistent with the size of the protein predicted from the sequence of COX11. The COX11 protein is tightly associated with the mitochondrial membrane but is not a component of purified cytochrome oxidase. An analysis of cytochrome oxidase subunits in wild type and in a cox11 mutant suggests that the COX11 protein is not required either for synthesis or transport of the subunit polypeptides into mitochondria. It seems more probable that COX11 protein exerts its effect at some terminal stage of enzyme synthesis, perhaps in directing assembly of the subunits.

Amino Acid Sequence↗

PubMed1989

Role of supernumerary subunits in mitochondrial cytochrome c oxidase.

Role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the influence on the enzymatic activity of their removal by chromatographic procedures or controlled digestion by trypsin. Is has been shown that partial proteolytic cleavage of subunit IV results in depression of respiratory activity and of redox-linked proton translocation. Selective removal by gel-filtration of subunit Vlb has no significant influence on the redox and protonmotive activity of the oxidase.

Animals↗

PubMed1989

Influence of surface charge on the incorporation and orientation of cytochrome c oxidase in liposomes.

Cytochrome c-oxidase is usually oriented 80-90% right-side-out when reconstituted with asolectin by the cholate dialysis method. Transformation of positively charged lysine groups at the matrix domain into negatively charged groups with succinic anhydride results in random orientation. A random orientation is also found after reconstitution in phosphatidylcholine, which can be changed into predominant right-side-out orientation by addition of cardiolipin. It is concluded that electrostatic interaction between positively charged groups of cytochrome c-oxidase with negative groups of phospholipids determines the asymmetric orientation of the enzyme in liposomes.

Acetic Anhydrides↗

PubMed1988

Characteristics of the protonmotive activity of mammalian cytochrome c oxidase and their modification by amino acid reagents.

Experimental analysis of the protonmotive activity of reconstituted cytochrome c oxidase from beef heart reveals the following features: (1) The observed H+:e- ratio for redox-linked proton ejection from oxidase vesicles is variable, being affected by various effectors that also influence the catalytic process. (2) Proton ejection appears to be associated with electron transfer from heme a (+CuA) to heme a3 (+CuB). (3) Chemical modification studies contribute to indentification of proton-conduction pathways in the protein and/or residues involved in the coupling process between redox and protonmotive activity. In intact rat liver mitochondria, under physiological conditions of dehydrogenase activity and delta microH+ generation by the respiratory chain cytochrome oxidase, does not appear to contribute significant H+ pumping. The relevance of what is observed is discussed in terms of possible mechanisms and physiological role.

Animals↗

PubMed1987

Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes.

In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.

Animals↗

PubMed1983

Characteristics of redox-linked proton ejection in cytochrome c oxidase reconstituted in phospholipid vesicles. New observations support mechanisms different from proton pumping.

Experimental observations reveal a number of characteristics of the redox-linked proton ejection from cytochrome c oxidase vesicles, which apparently cannot be explained by a proton pumping activity of the oxidase. These observations seem, on the other hand, to provide useful elements for alternative explanation(s) of the proton ejection. It is proposed here that the process is scalar and not vectorial and can derive from redox-linked rupture of protonated salt-bridges in the oxidase-lipid complex.

Biological Transport, Active↗

PubMed1983

Proton-cation translocation in tumor cell mitochondria.

The capacity of mitochondria isolated from tumor cells to conserve the transmembrane electrochemical proton gradient set up by respiration has been studied. In a K+ medium, mitochondria from Ehrlich ascites tumor cells exhibit a capacity to conserve aerobic delta microH comparable to that displayed by normal rat liver mitochondria. Mitochondria from Morris hepatoma 3924A show a decreased capacity to store delta microH+, which is principally due to lowering of delta pH. In a Na+ medium, both species of tumor mitochondria show a significant decrease of aerobic delta pH, while delta psi is the same, with respect to rat liver mitochondria. Experiments on passive swelling show that mitochondria from ascites tumor cells have an enhanced permeability to chloride salts of monovalent cations and increased activity of the Na+ (K+)-H+ exchange system of the mitochondrial membrane with respect to normal mitochondria. The enhanced activity of this system in ascites cells is also shown by the characteristics of respiration-linked proton translocation in submitochondrial particles and subsequent anaerobic proton diffusion. It is concluded that the decreased capacity of mitochondria from tumor cells to conserve aerobic delta pH is due to enhanced cyclic flow of Na+ across the membrane.

Aerobiosis↗

PubMed1980

The H+/e- stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria.

1. The -->H(+)/e(-) quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O(2), ferricyanide or ferricytochrome c, and from NNN'N'-tetramethyl-p-phenylenediamine+ascorbate to O(2), were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10(-3) pH unit. 3. The -->H(+)/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O(2) consumption or duroquinol oxidation. Higher -->H(+)/O quotients for succinate oxidation, obtained from polarographic measurements of O(2) consumption, resulted from underestimation of the respiratory rate. 4. The -->H(+)/2e(-) quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the -->H(+)/e(-) ratio was only 0.25 and not 0.7-1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the -->H(+)/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.

Animals↗

PubMedDate not supplied

Characteristics and nature of redox-linked proton transfer reactions in cytochrome c oxidase of mitochondria.

A discussion is presented of the characteristics of proton transfer reactions associated to redox catalysis in mitochondrial cytochrome c oxidase. These properties are examined in the light of the mechanisms proposed for the conversion of redox energy into a transmembrane proton gradient. It is concluded that this energy transfer process is first of all due to the anisotropic arrangement of the reduction of oxygen to H2O in the oxidase. The experimental observations available seem, on the other hand, to raise doubts on the capacity of cytochrome oxidase to function under steady-state conditions, in the native membrane, as a proton pump.

Antimycin A↗

PubMedDate not supplied

Regulation by the cAMP cascade of oxygen free radical balance in mammalian cells.

A study is presented of the effect of the cAMP cascade on oxygen metabolism in mammalian cell cultures. Serum-starvation of the cell cultures resulted in depression of the forward NADH-ubiquinone oxidoreductase activity of complex I, decreased content of glutathione, and enhancement of the cellular level of H2O2. Depressed transcription of cytosolic Cu/Zn-SOD 1, mitochondrial glutathione peroxidase and catalase was also observed. Activation of the cAMP cascade reversed the depression of the activity of complex I and the accumulation of H2O2. The effect of cAMP involved the cAMP-dependent protein kinase.

Animals↗

PubMedDate not supplied

The study of gap junctional intercellular communication in keratinocytes as screening of promoter effect induced by industrial and environmental toxic substances.

BACKGROUND: Disordered functioning of gap junctions between normal and initiated cells has been proposed as one possible mechanism of tumour promotion. Many putative carcinogens such as peroxisome proliferators, are known to activate various signal transduction mechanisms and modulate gap junctional intercellular communication (GJIC). They act as tumour promoters on pre-existing "initiated" cells, rather than as genotoxic initiators. OBJECTIVES: The aim of this article is to provide a screening-tool to evaluate the promoter carcinogen effect of environmental and occupational chemical contaminants, focusing on their ability to alter GJIC. METHODS: GJIC was investigated in serum-free cultured primary human keratinocytes, by directly evaluating the intercellular transfer of a microinjected fluorescent dye (Dye transfer). The expression of caspase 3, which is the ultimate target to be activated of both mitochondrial- and non-mitochondrial-linked pro-apoptotic pathways, was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: Mercury chloride (10 nM), mono-methyl Mercury (250 nM) and Trichloroethylene (500 I1M) were shown to significantly inhibit GJIC. Conversely di-methyl mercury, lead acetate and epichloridine had no effect on GJIC. All Trans Retinoic Acid completely reversed the inhibitory effect on GJIC induced by HgCI2 but not that induced by mono-methyl mercury and trichloroethylene. The result of a RT-PCR assay on total RNA cell extract showed that treatment of keratinocytes with 10 nM HgCl2 resulted in a decrease of the pro-apoptotic caspase 3 expression. CONCLUSIONS: In this work a protocol is designed to study gap junction intercellular communication in primary cultures of human keratinocytes which could be used as a reliable screening tool to test the promoter carcinogen effect of various environmental and occupational contaminants.

Air Pollutants, Occupational↗