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R Fato

Publications and source records attributed to R Fato.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1993

Gingival fibroblasts "in vitro" and Down's syndrome.

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of chromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to in using fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.

Adult↗
PubMed1992

Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria.

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.

Animals↗
PubMed1992

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria.

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochrome c oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochrome c reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochrome c oxidoreductase is strongly stimulated by added CoQ3, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of the Km for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14 mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ9 and CoQ10.

Adipose Tissue, Brown↗
PubMed1992

Localization and preferred orientations of ubiquinone homologs in model bilayers.

The localization of ubiquinone has been investigated in phospholipid bilayer vesicles in studies of fluorescence quenching of membrane-bound probes by ubiquinone homologs (Qn, where n is the number of the isoprenoid units of the chain). Fluorescence-quenching data obtained by using a set of anthroylstearate probes, having the fluorophore located at different depths, revealed that ubiquinone-3 is located throughout the whole bilayer thickness. From the bimolecular quenching constants in the membrane, lateral diffusion coefficients in two dimensions were calculated to span values of 10(-7)-10(-6) cm2.s-1. This suggests that ubiquinones laterally diffuse in a very fluid environment. On this basis, it is proposed that their translational diffusion in the bilayer takes place in two dimensions, with the quinone ring oscillating between the two bilayer surfaces within a hydrophobic environment not extending beyond the glycerol region. This model implies that the quinonic head is both settled near the polar surface of the bilayer and buried into the host hydrocarbon interior. This two-site distribution was confirmed for all Qn, except Q0, by their linear dichroism spectra in the bilayers provided by disc-like lyotropic nematic liquid crystals. These spectra also provided detailed information on the preferential orientations of the quinonic head of the different derivatives within the two sites. The mechanism by which the localization and orientation of Qn guest molecules inside the host bilayer is modulated by the isoprenoid chain length is discussed on a thermodynamical basis. Being that Qn is expected to be also widely contained in the highly curved cristae of the mitochondrial inner membrane, by using rod-like lyotropic nematic liquid crystals we searched out effects of the curvature of the host bilayer on those Qn distributions. The linear dichroism measurements reveal that Qn guest molecules are no longer obliged to find a partition between two different types of localizations when the host bilayer is highly curved. In this case all Qn, even the longest Q10, were found to stay parallel to the amphiphilic chains with a single site localization of the head near the polar interface. By the same linear dichroism technique, the local ordering of all Qn derivatives was also evaluated. The order parameters were found to be basically the same for all derivatives. This result is justified on the basis of the relaxation, caused by the surface curvature, of the lateral compression of the host chains.

Chemical Phenomena↗
PubMed1990

Studies on the role of ubiquinone in the control of the mitochondrial respiratory chain.

This study examines the possible role of Coenzyme Q (CoQ, ubiquinone) in the control of mitochondrial electron transfer. The CoQ concentration in mitochondria from different tissues was investigated by HPLC. By analyzing the rates of electron transfer as a function of total CoQ concentration, it was calculated that, at physiological CoQ concentration NADH cytochrome c reductase activity is not saturated. Values for theoretical Vmax could not be reached experimentally for NADH oxidation, because of the limited miscibility of CoQ10 with the phospholipids. On the other hand, it was found that CoQ3 could stimulate alpha-glycerophosphate cytochrome c reductase over three-fold. Electron transfer being a diffusion-coupled process, we have investigated the possibility of its being subjected to diffusion control. A reconstruction study of Complex I and Complex III in liposomes showed that NADH cytochrome c reductase was not affected by changing the average distance between complexes by varying the protein: lipid ratios. The results of a broad investigation on ubiquinol cytochrome c reductase in bovine heart submitochondrial particles indicated that the enzymic rate is not diffusion-controlled by ubiquinol, whereas the interaction of cytochrome c with the enzyme is clearly diffusion-limited.

Adipose Tissue↗
PubMed1990

Coenzyme Q can control the efficiency of oxidative phosphorylation.

Energy of metabolic oxidations is conserved in the form of ATP by the process of oxidative phosphorylation in mitochondria. The possibility to recognize alterations in the efficiency of oxidative phosphorylation in pathological states and to improve this efficiency in order to correct diseases requires knowledge of the mechanisms controlling the rate of ATP synthesis. This task is hampered by uncertainties still existing on the organization and mechanism of the enzymes carrying out oxidative phosphorylation. The authors have collected experimental evidence that coenzyme Q concentration in the mitochondrial membrane phospholipids in physiologically not saturating for maximal electron-transfer rate: in fact the Km of the redox enzymic complexes, using the oxidized and reduced form of coenzyme Q, for these substrates, are in the range of their concentrations in the membrane. The addition of exogenous coenzyme Q enhances the respiratory turnover above the physiological rate but without reaching theoretical Vmax, owing to the limited miscibility of ubiquinones with the membrane phospholipids. On the contrary, a decrease of ubiquinone content in the membrane lowers electron-transfer activity in a reversible fashion. Taking account that the rate of lateral coenzyme Q diffusion in the membrane does not appear to control electron transfer, it is suggested that only ubiquinone concentration affects the efficiency of oxidative phosphorylation, with interesting pathological and pharmacological implications.

Animals↗
PubMed1988

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles.

The steady-state kinetics of ubiquinol cytochrome c reductase was investigated in submitochondrial particles using ubiquinol-1 as electron donor in media of increasing viscosities obtained by water-polyethylene glycol mixtures. The minimum association rate constant, kmin = kcat/km, for cytochrome c was strongly viscosity dependent, whereas kmin for ubiquinol-1 was only weakly affected by viscosity. It is concluded that the interaction of cytochrome c with the membranous reductase is largely under diffusion control, whereas the oxidation of ubiquinol by the enzyme is not significantly controlled by diffusion in either the aqueous medium or the membrane. The results are compatible with the presence of a diffusion limited step in cytochrome c but not in ubiquinone in mitochondrial electron transfer.

Animals↗
PubMed1988

A simple method for the determination of the kinetic constants of membrane enzymes utilizing hydrophobic substrates: ubiquinol cytochrome c reductase.

We have devised a method to determine the true Km of membrane enzymes for hydrophobic substrates dissolved in lipid bilayers, and the lipid/water partition coefficients, by simple steady-state kinetic measurements at varying membrane phospholipid fractional volumes in the assay medium. The method has been applied to mitochondrial ubiquinol cytochrome c reductase, using short-chain ubiquinols as reductants at saturating cytochrome c. The partition coefficients of the quinols, as obtained by this method, are in good agreement with those determined directly by other procedures; Km values obtained by this method, when expressed as concentrations in the lipid bilayer, are in the millimolar range. The kinetics of the ubiquinol analog duroquinol are independent of phospholipid concentration, as expected from its partition coefficient close to unity.

Algorithms↗
PubMed1986

Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes.

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Is ubiquinone diffusion rate-limiting for electron transfer?

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, random distribution as above, but with ubiquinone diffusion not rate-limiting, diffusion and collision of protein complexes carrying bound ubiquinone, and solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 X 10(9) M-1 sec-1 corresponding to a diffusion coefficient in the range of 10(-6) cm2/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G., Biochemistry, 25, 3378-3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R., Proc. Natl. Acad. Sci. USA 81, 2606-2610, 1984) is 3 X 10(-9) cm2/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochrome c reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochrome c reductase or succinate-cytochrome c reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below the Km of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization.(ABSTRACT TRUNCATED AT 400 WORDS)

Diffusion↗
PubMed1985

Measurement of the lateral diffusion coefficients of ubiquinones in lipid vesicles by fluorescence quenching of 12-(9-anthroyl)stearate.

The lateral diffusion coefficients of some ubiquinone homologues have been measured in phospholipid vesicles exploiting the fluorescence quenching of the probe 12-(9-anthroyl)stearate by the quinones. Diffusion coefficients higher than 10(-6) cm2 X s-1 have been found at 25 degrees C, compatible with the localization of the ubiquinones in the low-viscosity midplane region of the bilayer.

Diffusion↗
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

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↗
PubMed1983

A temperature-dependent structural change of mitochondrial ATPase.

The temperature dependence of the intrinsic tryptophan fluorescence in either bovine heart submitochondrial particles or oligomycin-sensitive ATPase isolated therefrom shows a discontinuity at near 25 degrees C, which coincides with the temperature where a break in the Arrhenius plot of ATPase activity is found. Addition of n-butanol to submitochondrial particles induces a decrease of tryptophan fluorescence in the whole temperature range. The discontinuity is interpreted as a temperature-dependent structural change and related to a viscosity-induced phase separation of the intrinsic mitochondrial proteins.

1-Butanol↗
PubMed1980

Effect of some lipophilic substances on fluorescence polarization of perylene in lipid vesicles and mitochondrial membranes.

Short-chain ubiquinones were observed to increase the fluorescence polarization associated with perylene, indicating a decrease in the fluidity of the mitochondrial membrane. The results indicated that the perturbation induced by low homologs results indicated that the perturbation induced by low homologs of ubiquinone on the physical state of membrane lipids is quite different from that of other lipophilic substances which have been considered.

Animals↗