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Biomedical subjects

G Lenaz

Publications and source records attributed to G Lenaz.

At least 127 records · Page 7Linked to original sources
PubMed1985

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrial bc1 complex.

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrial bc1 complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in the bc1 complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochrome c reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of the bc1 complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochrome c reductase activity. However, a clear stimulation of the rate of cytochrome b reduction in parallel to an inhibition of cytochrome b oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on the bc1 complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.

Animals↗
PubMed1985

Effect of ubiquinone extraction on the reaction of the mitochondrial bc1 complex with ferricyanide.

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochrome b extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochrome c was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/mol bc1 complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochrome b extrareduction. Since the initial rates of cytochrome b reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochrome b was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.

Animals↗
PubMed1985

The essentiality of coenzyme Q for bioenergetics and clinical medicine.

Coenzyme Q is an essential component of the respiratory chain, where it represents a mobile pool between dehydrogenases and cytochromes. The fact that Q is a free component, and its concentration is not in great excess over the Km of the respiratory complexes, renders this compound potentially rate-limiting in the respiratory chain. On the other hand, the rate of lateral diffusion of Q in the mitochondrial membrane is not a limiting step under physiological conditions. Quinoid compounds, which act as inhibitors of the respiratory chain at the level of Q, besides being useful tools for investigations of electron transfer, could be important in pathology as inhibitors of respiration.

Animals↗
PubMed1984

Effects of dibromothymoquinone on the structure and function of the mitochondrial bc1 complex.

We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).

Animals↗
PubMed1984

Fluidizing effect of endogenous ubiquinone in bovine heart mitochondrial membranes.

Extraction of endogenous ubiquinone from lyophilized beef heart mitochondria results in increases of both the order parameter of the spin label 5-NS and of the rotational correlation time of 16-NS; reconstitution with the pentane extract results in restoration of the original spectral parameters. On the other hand, addition of purified ubiquinone homologs restores the original spectra only in the case of 16-NS, whereas the order parameter of 5-NS is restored by addition of mixed phospholipids. The same amounts of ubiquinone homologs incorporated in mixed phospholipid vesicles induce much lower effects. It is suggested that ubiquinone in mitochondria is intercalated with the lipid chains of the membrane in such a way to perturb the fluidity of the hydrophobic core.

Animals↗
PubMed1984

On the mechanism of inhibition of NADH oxidase by ubiquinone-3.

The combined effects of rotenone and ubiquinone-3 on the kinetics of NADH dehydrogenase and NADH oxidase have been investigated. The two inhibitors do not show additivity; on the other hand, ubiquinone-3, when preincubated with the enzyme, partially removes rotenone sensitivity. The inhibition of NADH oxidase by ubiquinone-3 is the result of at least two combined effects: the competition of the less active ubiquinone-3 with endogenous ubiquinone-10 in the acceptor site of the dehydrogenase, and a nonspecific action on the structure of complex I. The latter effect is perhaps mediated by a physical change of the phospholipid bilayer similar to that observed with agents such as butanol, perturbing lipid-protein interactions in the membrane.

Animals↗
PubMed1984

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase.

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimyristoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18-28 degrees C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots of Km also show breaks in the same temperature interval, with the exception of the dioleoyl-enzyme vesicles, where Km is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24 degrees C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3-18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.

Animals↗
PubMed1983

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide.

N,N'-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c1, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c1 complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c1 complex. The binding of [14C]DCCD to the isolated b-c1 complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c1 complex are discussed.

Animals↗
PubMed1983

Inhibition of the mitochondrial bc1 complex by dibromothymoquinone.

We have studied the effects of dibromothymoquinone (DBMIB) in various redox activities of the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations higher than 50 mol/mol of cytochrome c1 the inhibitor produces a bypass of electron transfer on the substrate side of the bc1 complex, because of its autooxidation capability. This induces an artifactual overestimation of the real inhibition titer of the redox activity of this enzyme, which has been found to be 3-6 mol/mol of cytochrome c1 by following the ubiquinol-cytochrome c reductase activity. This action is reversed by addition of excess of sulphydryl compounds like cysteine.

Animals↗
PubMed1983

Temperature-dependent conformational changes in isolated oligomycin-sensitive ATPase.

Isolated oligomycin-sensitive ATPase undergoes a kinetic change at 20-25 degrees C with a higher activation energy and a lower Km for ATP below this temperature range. This observation has been correlated with temperature-dependent structural changes detected by circular dichroism in the UV region in the isolated enzyme. The negative ellipticities in the 208-225 nm region, which are proportional to the alpha-helix content, increase with rise in temperature to a maximum above 25 degrees C.

Adenosine Triphosphatases↗
PubMed1983

Electron spin resonance studies of the effects of lipids on the environment of proteins in mitochondrial membranes.

The physical state of mitochondrial membranes has been investigated by means of stearic acid spin labels and of a maleimide spin label covalently bound to protein sulfhydryl groups. Stearic acid spin labels 5-NS and 16-NS show that n-butanol enhances the lipid fluidity of mitochondrial membranes in the whole temperature range between 4 and 37 degrees C; the effects in the hydrophobic membrane core, probed by 16-NS, are already apparent at 10 mM butanol. In liposomes formed of mitochondrial phospholipids, a fluidizing effect appears only at much higher concentration. Such results are compatible with the idea that butanol destabilizes lipid-protein interactions. On the other hand, the ratio between weakly and strongly immobilized SH groups probed by maleimide spin label is only slightly affected in the temperature range of 4-37 degrees C by addition of high concentrations of n-butanol, indicating that the environments probed are stable to agents inducing fluidity changes in the lipids. There are, however, indications that the environment probed by maleimide is affected by lipids, since the spin label, when bound to lipid-depleted mitochondria, becomes more immobilized, reconstitution of such lipid-depleted membranes with phospholipids restores the original spectra.

Adenosine Triphosphatases↗
PubMed1982

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles.

A kinetic study on ubiquinol-cytochrome c reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c1 complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a Km for ubiquinol-1 of 13.4 microM in mitochondria and of 24.6 microM in the isolated cytochrome b-c1 complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent Km values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the Vmax and the Km for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.

Animals↗