Biomedical subjects

R Fato

Publications and source records attributed to R Fato.

At least 19 recordsLinked to original sources

PubMed1999

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.

Animals↗

PubMed1998

A high diffusion coefficient for coenzyme Q10 might be related to a folded structure.

We measured the lateral diffusion of different coenzyme Q homologues and analogues in model lipid vesicles using the fluorescence collisional quenching technique with pyrene derivatives and found diffusion coefficients in the range of 10(-6) cm2/s. Theoretical diffusion coefficients for these highly hydrophobic components were calculated according to the free volume theory. An important parameter in the free volume theory is the relative dimension between diffusant and solvent: a molecular dynamics computer simulation of the coenzymes yielded their most probable geometries and volumes and revealed surprisingly similar sizes of the short and long homologues, due to a folded structure of the isoprenoid chain in the latter, with a length for coenzyme Q10 of 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s, in good agreement with those found experimentally.

Coenzymes↗

PubMed1997

Steady-state kinetics of reduction of coenzyme Q analogs by glycerol-3-phosphate dehydrogenase in brown adipose tissue mitochondria.

We have undertaken a study of the role of coenzyme Q (CoQ) in glycerol-3-phosphate oxidation in mitochondrial membranes from hamster brown adipose tissue, using either quinone homologs, as CoQ1 and CoQ2, or the analogs duroquinone and decylubiquinone as artificial electron acceptors. We have found that the most suitable electron acceptor for glycerol-3-phosphate:CoQ reductase activity in situ in the mitochondrial membrane is the homolog CoQ1 yielding the highest rate of enzyme activity (225 +/- 41 nmol x min(-1) x mg(-1) protein). With all acceptors tested the quinone reduction rates were completely insensitive to Complex III inhibitors, indicating that all acceptors were easily accessible to the quinone-binding site of the dehydrogenase preferentially with respect to the endogenous CoQ pool, in such a way that Complex III was kept in the oxidized state. We have also experimentally investigated the saturation kinetics of endogenous CoQ (1.35 nmol/mg protein of a mixture of 70% CoQ9 and 30% CoQ10) by stepwise pentane extraction of brown adipose tissue mitochondria and found a K(m) of the integrated activity of glycerol-3-phosphate cytochrome c reductase for endogenous CoQ of 0.22 nmol/mg protein, indicating that glycerol-3-phosphate-supported respiration is over 80% of V(max) with respect to the CoQ pool. A similar K(m) of 0.19 nmol CoQ/mg protein was found in glycerol-3-phosphate cytochrome c reductase in cockroach flight muscle mitochondria.

Adipose Tissue, Brown↗

PubMed1997

Saturation kinetics of coenzyme Q in NADH oxidation: rate enhancement by incorporation of excess quinone.

In beef heart mitochondria it has been found that the Km for coenzyme Q10 of the NADH oxidation system is in the range of the membrane concentration of the quinone; this is contrary to succinate oxidation which is in Vmax with respect to quinone content. The same proportional difference between the two systems is maintained in their affinities for the exogenous acceptor CoQ1 in non-extracted mitochondria. The Km of succinate- coenzyme Q reductase for CoQ1 is reversibly lowered in CoQ-depleted mitochondria; while in contrast the Km for NADH-coenzyme Q reductase is reversibly increased by CoQ extraction. Incorporation of exogenous quinones by co-sonication with submitochondrial particles, as evidenced by fluorescence quenching of pyrene, enhances NADH-cytochrome c reductase activity in accordance with the lack of saturation of the former system.

Animals↗

PubMed1997

The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes.

The experiments reported here were undertaken to test the hypothesis that the antioxidative, reduced form of hydrophobic phase coenzyme Q (CoQ) may be generated and maintained by the two-electron quinone reductase, DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] by catalyzing formation of the hydroquinone form of CoQ. This enzyme was isolated and purified from rat liver cytosol and its reduction of several CoQ homologs incorporated into large unilamellar vesicles (LUVETs) was demonstrated. The addition of NADH and DT-diaphorase to LUVETs and to multilamellar vesicles (MLVs) containing CoQ homologs, including CoQ9 and CoQ10, resulted in essentially complete reduction of the CoQ. Incorporation of either CoQ9H2 or CoQ10H2 and the lipophylic radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) into MLVs in the presence of DT-diaphorase and NADH maintained the reduced state of CoQ and inhibited lipid peroxidation. The reaction between DT-diaphorase and CoQ was also demonstrated in isolated rat liver hepatocytes in which incorporation of CoQ10 provided protection from adriamycin (adr)-induced mitochondrial membrane damage. The role of DT-diaphorase in the antioxidant activity of CoQ was demonstrated by the co-incorporation of dicoumarol (dic), a potent inhibitor of DT-diaphorase, resulting in a loss of protection by incorporated CoQ10. These results support the antioxidant function of DT-diaphorase in both artificial and natural membrane systems by acting as a two-electron CoQ reductase which forms and maintains CoQ in the reduced state.

Animals↗

PubMed1997

Mitochondrial complex I defects in aging.

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed. The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase. A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats. In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases.

Adult↗

PubMed1996

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

Animals↗

PubMed1996

Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles.

The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6-pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ2. The Kmin (kcat/Km) for CoQ1 is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of kmin is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kröger, A., & Klingenberg, M. (1973) Eur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool.

Animals↗

PubMed1995

Major changes in complex I activity in mitochondria from aged rats may not be detected by direct assay of NADH:coenzyme Q reductase.

We have investigated the respiratory activities and the concentrations of respiratory chain components of mitochondria isolated from the livers and hearts of two groups of rats aged 6 and 24 months respectively. In comparison with the adult controls (6 months), in aged rats there was a decline in total aerobic NADH oxidation in both tissues; only minor (non-significant) changes, however, were found in NADH:coenzyme Q reductase and cytochrome oxidase activities, and there was no change in ubiquinol-cytochrome c reductase activity. The coenzyme Q levels were slightly decreased in mitochondria from both organs of aged rats. The lowered NADH oxidase activity is not due to the slight decrease observed in the coenzyme Q levels, but is the result of decreased Complex I activity. Since the assay of NADH:coenzyme Q reductase requires quinone analogues, none of which can evoke its maximal turnover [Estornell, Fato, Pallotti and Lenaz (1993) FEBS Lett. 332, 127-131], its activity has been calculated indirectly by taking advantage of the relationship that exists between NADH oxidation and ubiquinol oxidation through the coenzyme Q pool. The results, expressed in this way, show a drastic loss of activity of Complex I in both the heart and the liver of aged animals in comparison with adult controls.

Aging↗

PubMed1995

Underevaluation of complex I activity by the direct assay of NADH-coenzyme Q reductase in rat liver mitochondria.

We have shown that the rate of NADH-coenzyme Q reductase in rat liver mitochondria, assayed using the decyl-ubiquinone analog DB, is underevaluated, probably as a result of its low water solubility. In view of drawbacks encountered using other more soluble acceptors in this system, we demonstrate that the most reliable assay of the physiological rate of CoQ reduction by Complex I is the indirect calculation from the total rate of NADH oxidation and the rate of ubiquinol oxidation, using the pool equation of Kröger and Klingenberg [(1973) Eur. J. Biochem. 34, 358-368].

Animals↗

PubMed1994

Mitochondrial activities of rat heart during ageing.

Some analytical and functional parameters of rat heart mitochondrial have been investigated at six different periods of ageing from 2 to 26 months. The fatty acid composition of the mitochondrial membranes reveals a percentage increase of polyunsaturated fatty acids (20:4 n-6, 22:6 n-3) up to 12 months, followed by a decrease; however, fluorescence polarization of the membrane probe diphenylhexatriene is not changed, revealing that membrane fluidity is not significantly affected. No major change in ubiquinone-9 and in cytochrome content is apparent, indicating that the relative ratio of the respiratory chain components is unmodified. Nevertheless, significant changes in enzyme specific activities are detected: NADH cytochrome c reductase and cytochrome oxidase activities increase up to 12 months, then decrease at 18-26 months; ubiquinol cytochrome c reductase exhibits a peak at 18 months, followed by a decrease. All these activities follow a similar trend during the whole life span of the rat, even though the 'maximum' is different. No significant changes have been found in ATP synthase. Succinate-cytochrome c reductase steadily increases over the whole life span. The results, showing activity decreases in the respiratory enzymes having subunits encoded by mitochondrial DNA, are compatible with the 'mitochondrial' theory of ageing.

Aging↗

PubMed1994

An updating of the biochemical function of coenzyme Q in mitochondria.

The apparent Km for coenzyme Q10 in NADH oxidation by coenzyme Q (CoQ)-extracted beef heart mitochondria is close to their CoQ content, whereas both succinate and glycerol-3-phosphate oxidation (the latter measured in hamster brown adipose tissue mitochondria) are almost saturated at physiological CoQ concentration. Attempts to enhance NADH oxidation rate by excess CoQ incorporation in vitro were only partially successful: the reason is in the limited amount of CoQ10 that can be incorporated in monomeric form, as shown by lack of fluorescence quenching of membrane fluorescent probes; at difference with CoQ10, CoQ5 quenches probe fluorescence and likewise enhances NADH oxidation rate above normal. Attempts to enhance the CoQ content in perfused rat liver and in isolated hepatocytes failed to show uptake in the purified mitochondrial fraction. Nevertheless CoQ cellular uptake is able to protect mitochondrial activities. Incubation of hepatocytes with adriamycin induces loss of respiration and mitochondrial potential measured in whole cells by flow cytometry using rhodamine 123 as a probe: concomitant incubation with CoQ10 completely protects both respiration and potential. An experimental study of aging in the rat has shown some decrease of mitochondrial CoQ content in heart, and less in liver and skeletal muscle. In spite of the little change observed, it is reasoned that CoQ administration may be beneficial in the elderly, owing to the increased demand for antioxidants.

Aging↗

PubMed1994

Protective effect of exogenous coenzyme Q against damage by adriamycin in perfused rat liver.

We have investigated the effect of rat liver perfusion with adriamycin on mitochondrial activities. Although the perfusion treatment per se induces some decline of respiratory activities, adriamycin strongly potentiates this effect; moreover the coenzyme Q9 content of the mitochondrial membrane is significantly lowered by the antibiotic. Coaddition of coenzyme Q10 in the perfusate significantly protects the mitochondria, not only from loss of respiratory activities but also of the endogenous CoQ9 content. Exogenous CoQ10 fails to enhance respiratory activities in control rats, not treated with adriamycin, even though CoQ concentration has been proven not to be kinetically saturating in the respiratory chain under physiological conditions. Thus, the beneficial effect of CoQ10 in the perfusate does not appear to be the result of its role in the respiratory chain but is a consequence of its antioxidant action.

Animals↗

PubMed1993

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase.

The assay of Complex I activity requires the use of artificial acceptors, such as short-chain coenzyme Q homologs and analogs, because the physiological quinones, such as CoQ10, are too insoluble in water to be added as substrates to the assay media. The medical interest raised in the last years on the pathological changes of Complex I activity has focussed on the requirement of easy reliable assays for its analysis. We have undertaken a systematic examination of the assay conditions of Complex I in mitochondrial membranes, using a series of quinones as electron acceptors, particularly the coenzyme Q homologs CoQ0, CoQ1 and CoQ2, and the analogs duroquinone and decylubiquinone. Our findings have pointed out that the most suitable electron acceptor for the NADH:CoQ reductase assay is the homolog CoQ1. The analog DB, commercially available, although yielding a high activity, nevertheless causes some problems for the standardization of the assay conditions.

Animals↗

PubMed1993

Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists.

Mitochondrial complex I and complex III have common inhibitors with ubiquinone-like structure. The tridecyl analog of stigmatellin, which inhibits mitochondrial complex III at nanomolar concentrations, also inhibits the NADH:ubiquinone reductase activity of complex I at micromolar concentrations. The inhibitor titer depends upon the concentration of the mitochondrial particles and extrapolates to 0.2 microM at zero particle concentration. The stigmatellin analog is more powerful than its parent compound and is noncompetitive with exogenous ubiquinones, rotenone and piericidin. Myxothiazol, which is another potent inhibitor of complex III, is also found to inhibit the activity of complex I with a titer comparable to that of the tridecyl analog of stigmatellin. Additionally, piericidin, which is the most powerful inhibitor of complex I, inhibits the ubiquinol:cytochrome c reductase activity of complex III at micromolar concentrations in mitochondrial particles and at submicromolar concentrations in the isolated enzyme complex.

Animals↗

PubMed1993

Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects.

In an attempt to establish the relative importance of diffusional and chemical control in the reactivity of the two of the two substrates, ubiquinol and cytochrome c, we have undertaken as extensive characterization of the steady-state kinetics of ubiquinol-cytochrome c reductase (EC 1.10.2.2) when present in open submitochondrial particles from bovine heart. The kinetic pattern follows a Ping Pong mechanism; contrary to the situation found with the isolated enzyme [Speck and Margoliash (1984) J. Biol. Chem. 259, 1064-1072, and confirmed in our laboratory], no substrate inhibition by oxidized cytochrome c was observed with the membrane-bound enzyme. Endogenous oxidized ubiquinone-10 is unable to exert product inhibition under the conditions employed. In the Ping Pong mechanism for this enzyme, the reaction scheme can be clearly divided into two parts, and the Kmin. (kcat./km) value for one substrate is independent of the rate constant for the second substrate. Both ubiquinol-1 and ubiquinol-2 can be used as electron donors reacting with the enzyme from within the lipid bilayer [Fato, Castelluccio, Palmer and Lenaz (1988) Biochim. Biophys. Acta 932, 216-222]; the kmin. values for ubiquinols, when calculated on the basis of their membranous concentrations, are significantly lower than the kmin. for cytochrome c. The temperature-dependence of the kinetic parameters was investigated by titrating each of the substrates under quasi-saturating concentrations of the second substrate. Arrhenius plots of Vmax. extrapolated from both cytochrome c and ubiquinol titrations were linear, when care was taken to verify the quasi-saturating concentrations of the fixed co-substrate. The Arrhenius plots for the kmin. values for both ubiquinol and cytochrome c were linear, but the activation energy was much higher for the former, particularly when calculated for ubiquinol dissolved in the lipid phase; the very low value of activation energy of the kmin. for cytochrome c is strong support for diffusion control being present in the reaction of cytochrome c with the membranous enzyme. In contrast to the soluble enzyme, ubiquinone titrations of submitochondrial particles at low cytochrome c concentrations deviated from hyperbolic behaviour. Changing the medium viscosity with either poly(ethylene glycol) or sucrose had a strong effect on the cytochrome c kmin., whereas the change in the ubiquinol kmin. was much smaller. From the viscosity studies the extent of diffusional control could be calculated, revealing that the reaction with cytochrome c was mostly diffusion-limited. The viscosity of the membrane was changed by incorporating cholesterol; no significant effect on the ubiquinol kmin. ascribable to diffusion control could be recognized.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Steady-state kinetics of ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae mitochondria: effects of fluidity changes obtained by different growth temperatures.

We have investigated the steady-state kinetics of ubiquinol-cytochrome c reductase using either ubiquinol-1 or -2 as the donor substrate in mitochondria from Saccharomyces cerevisiae either grown at 36 degrees C or brought from 36 to 10 degrees C at the end of the exponential phase ("thermal shock"). The fatty acid composition of the mitochondrial lipids varied under the two conditions, with an increase of the unsaturation degree in the thermally shocked cells. The fluorescence polarization of the membrane probe diphenylhexatriene incorporated into mitochondria was decreased in a wide temperature range in the thermally shocked cells, in accordance with the fatty acid changes. Also the motion and order parameters of two fatty acid spin labels revealed significant changes in the thermally shocked cells, interpreted as an increase of membrane fluidity brought about by the thermal shock. This increase agrees with the finding that the diffusion coefficient of the ubiquinone homolog Q3 in the membrane, investigated by collisional fluorescence quenching of the membrane probe 12-(9-anthroyl) stearic acid, was consistently increased in the mitochondria from the thermally shocked cells. In spite of the physical changes of the membrane and the increase in the diffusion coefficients of the ubiquinone homolog, the steady-state kinetics of ubiquinol cytochrome c reductase showed no change to be interpreted in terms of a diffusion-limited component. An increase of Vmax of the enzyme in the thermally shocked cells was the result of an increased content of the bc1 complex; both the Km for ubiquinols and the turnover number were almost identical under the two growth conditions, so that the kcat/Km ratio, which approaches the second-order rate constant of substrate interaction with the enzyme active site, was unmodified. This is a demonstration that the ubiquinol cytochrome c reductase activity in S. cerevisiae mitochondria is not diffusion-controlled in the interaction of the donor substrate with the enzymatic protein.

Cytochromes↗

PubMed1993

The function of coenzyme Q in mitochondria.

We have accumulated evidence that coenzyme Q (CoQ) concentration in the mitochondrial membrane is not saturating for NADH oxidation but is saturating for succinate and glycerol-3-phosphate oxidation. As a result of its kinetic properties CoQ concentration changes must yield changes in respiration rates. This provides a rationale for the reported therapeutic effects of CoQ under conditions when its concentration is decreased, as has been reported in tissues from aged rats; we have failed, however, to detect any specific CoQ decrease in mitochondria from several tissues of aged rats. We can, however, predict from the kinetic bases that CoQ would ameliorate respiration rate also under conditions in which a defect is present in regions not involving the quinone. CoQ incorporation in perfused liver is attempted in order to find experimental systems for investigating its protecting effect. Liposomal CoQ10 perfused in rat livers (where CoQ9 is the main homolog) is incorporated mainly in lysosomes, and its increase in the crude mitochondrial fraction could be mainly ascribed to residual lysosomal contamination. Nevertheless, perfusion with exogenous CoQ10 maintains higher levels of endogenous CoQ9, and higher glutamate oxidation than in controls. In the same system, an oxidative stress by doxorubicin induces mitochondrial changes, including a decrease in endogenous CoQ9 and in respiratory activities. These changes are prevented by concomitant perfusion of liposomal CoQ10.

Aging↗