Biomedical subjects

G Lenaz

Publications and source records attributed to G Lenaz.

At least 55 records · Page 3Linked to original sources

PubMed1997

Mitochondrial respiratory chain features after gamma-irradiation.

Radiation provokes damage to DNA but also to membrane and protein structure. Radiolysis is a tool used very often in the study of free radical biological effects and of scavenger molecules effectiveness. Nitroimidazoles have been demonstrated to enhance the radiation effects on biological structures. The studies we have performed on isolated mitochondria irradiated, with and without nitroimidazoles, at a radiation dose equal to LD90, indicate that this treatment is not able to affect the structural and functional features investigated (ubiquinone-10, fatty acids, respiratory cytochrome levels or membrane fluidity and respiratory enzymatic activities), suggesting that an involvement of such externally produced radicals on membrane damage is unlikely. Moreover it was ascertained that the mitochondrial redox activities do not take part into the intracellular nitroimidazole reduction.

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↗

PubMed1996

Inhibitor sensitivity of respiratory complex I in human platelets: a possible biomarker of ageing.

NADH-Coenzyme Q reductase was assayed in platelet mitochondrial membranes obtained from 19 pools of two venous blood samples from female young (19-30 years) individuals and 18 pools from aged ones (66-107 years). The enzyme activities were not significantly changed in the two groups, but a decrease of sensitivity to the specific inhibitor, rotenone, occurred in a substantial number of aged individuals. The results are in agreement with the predictions of the mitochondrial theory of ageing and may be used to develop a sensitive biomarker of the ageing process.

Adult↗

PubMed1996

Coenzyme Q depletion in rat plasma after partial hepatectomy.

Coenzyme Q content was monitored in blood plasma and regenerating liver mitochondria of hepatectomized rats, using as controls either sham-operated or non-operated animals. Mitochondrial CoQ9 content increased in sham-operated rats, whilst it was significantly lower in hepatectomized in comparison with non-operated animals at all considered times. On the other hand plasma CoQ9 levels dramatically decreased in hepatectomized animals, while strongly increased in sham-operated in comparison with non-operated rats. The quinone decrease in hepatectomized animals is likely to be due to the attainment of a rate-limiting step in CoQ biosynthesis.

Animals↗

PubMed1995

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Adenosine Triphosphatases↗

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↗

PubMed1995

Coenzyme Q content in synaptic and non-synaptic mitochondria from different brain regions in the ageing rat.

We investigated the Coenzyme Q (CoQ) content of different mitochondrial fractions [free mitochondria (FM), synaptic heavy (HM) and light mitochondria (LM)] from three brain areas (cortex, striatum, hippocampus) of rats at different ages. In rats from 2 to 26 months of age, we observed only small differences in total CoQ content (CoQ9 + CoQ10). In FM and LM fractions, values are very similar and appear to be much higher than in HM fractions. The CoQ10/CoQ9 ratios are much higher in brain mitochondria than in other organs, suggesting possible modifications of CoQ biosynthetic pathways in brain; nevertheless they appear to remain constant during ageing. CoQ9 and CoQ10 contents slowly decrease reaching their minimum in rats of 18 months of age, then increase in the older ages. Considering ageing as partially driven by a summation of free radical-mediated processes, we can hypothesize that damage occurring to biological structures in the first half of life might be followed by induction phenomena tending to re-establish the primitive levels of antioxidant molecules.

Aging↗

PubMed1995

A study of the mitochondrial F1-ATPase tryptophan phosphorescence at 273 K.

The bovine heart mitochondrial F1-ATPase complex exhibits an intrinsic tryptophan phosphorescence that can be used to monitor structural changes of the epsilon-subunit. The phosphorescence decay rate of F1 containing the tightly bound nucleotides increases upon addition of adenine nucleoside triphosphate in the presence of magnesium. The average phosphorescence lifetime of this enzyme preparation decreases from 10.2 to 7.8 ms upon Mg-ATP addition. Since increasing phosphorescence decay rate is related to increasing flexibility of proteins, Mg-ATP added to the F1-ATPase complex can enhance the flexibility of the protein structure surrounding the chromophore. Experiments carried out on F1 prepared with the three noncatalytic sites filled and the three catalytic sites vacant show a significant increase of the phosphorescence lifetime from 6.4 ms to 7.6 ms upon Mg-ATP addition. These results suggest that the mitochondrial F1-ATPase epsilon-subunit conformation senses differently the nucleoside triphosphate binding to catalytic or noncatalytic sites.

Adenosine Triphosphate↗

PubMed1995

Human skeletal muscle mitochondria in aging: lack of detectable morphological and enzymic defects.

We have investigated structural and functional properties of skeletal muscle mitochondria obtained from biopsies from young and old individuals. The morphometric analysis of muscle sections revealed a tendency to an increase of total area, numerical density and volume density of mitochondria in the aged. The enzymatic activities of NADH-Coenzyme Q reductase, succinate cytochrome c reductase, ubiquinol-cytochrome c reductase exhibited a high variability of specific activities without any correlation with age. Expression of the values as enzyme turnovers reduced the variability but was unable to reveal any age-dependent modification.

Adolescent↗

PubMed1995

Function of coenzyme Q in the cell: some biochemical and physiological properties.

Coenzyme Q (CoQ), a lipophilic substituted benzoquinone, is well known as a redox component of the mitochondrial and many bacterial respiratory chains. However, additional locations and roles have been recently discovered. CoQ is described as a redox component of extramitochondrial electron transport chains and it is a powerful antioxidant and a membrane stabilizer. Increasing evidence for the beneficial clinical effects of CoQ administration in senescence or in different disorders (e.g. cerebrovascular, muscular, neurogenic) may be explained by the multiple roles of CoQ in cells.

Aging↗

PubMed1994

Functional alterations of the mitochondrially encoded ND4 subunit associated with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease associated with point mutations in mitochondrial DNA. The most frequent of these mutations is the G-to-A substitution at nucleotide position 11,778 which changes an evolutionarily conserved arginine with a histidine at position 340 in subunit ND4 of NADH:ubiquinone reductase (respiratory complex I). We report that this amino acid substitution alters the affinity of complex I for the ubiquinone substrate and induces resistance towards its potent inhibitor rotenone in mitochondria of LHON patients. Such changes could reflect a substantial loss in the energy conserving function of NADH:ubiquinone reductase and thus explain the pathological effect of the ND4/11,778 mutation.

Amino Acid Sequence↗

PubMed1994

Dietary lipids and 5'-nucleotidase activity of rat cell plasma membranes.

Three groups of adult rats were fed black currant or olive oils or a 1:1 mixture of the two for three months. Feeding black currant oil diets, high in 18:2 (n-6), 18:3 (n-6), 18:3 (n-3), increased the heart and liver plasma membrane content of linoleic and arachidonic acid with a concomitant decrease of oleic acid. PUFA, n-3 and n-6 content and the bilayer lipid fluidity as examined by measuring the fluorescence anisotropy of diphenylhexatriene were not significantly affected. On the contrary, the 5'-nucleotidase of liver membrane of rats fed black currant diets was lower than that observed in membranes of liver from olive oil fed rats. Therefore it is concluded that PUFA and n-3/n-6 ratio as well as membrane fluidity do not influence the 5'-nucleotidase activity. It is suggested that the activity is sensitive to the amount of a specific fatty acid of the membrane (i.e., oleic or arachidonic acid) and/or to lipid supplementation which can influence the eicosanoids metabolism.

5'-Nucleotidase↗

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

Peroxidative extent and coenzyme Q levels in the rat: influence of physical training and dietary fats.

Sport practice is widely recognized as capable of producing peroxidative damages, even of severe intensity. Dietary manipulations can also modify membrane susceptibility to peroxidation. In previous experiments we found that, while dietary virgin olive oil successfully protects mitochondrial and microsomal membranes from endogenous, xenobiotics-induced peroxidation, dietary polyunsaturated oils lead to increased peroxidative levels. In the latter conditions, cell machinery tries to counteract the structural and functional changes which have occurred, by modulating enzyme activities and concentrations, by increasing biosynthesis of coenzyme Q and by mobilizing cholesterol. In the present study we hypothesized that combining these two aspects could give useful information on the membrane response to peroxidation phenomena that daily occur throughout the lifespan. Rats fed different dietary oils as only fat source underwent a carefully designed training program and were killed at different times following acute or chronic exercise. Results show that peroxidation related to chronic training and to an acute bout of exercise sum up with peroxidative effects induced by dietary factors. The above mentioned phenomena occurred simultaneously with increased tissue levels of coenzyme Q, possibly triggered within a physiological reactive antioxidant strategy.

Aging↗

PubMed1994

The effect of dietary lipid changes on the fatty acid composition and function of liver, heart and brain mitochondria in the rat at different ages.

A correlation between dietary lipids and cellular enzyme activities is a problem that has only been partially addressed by nutritionists. Therefore, changes in the fatty acid composition and the activities of some key metabolic enzymes (ubiquinol-2-cytochrome c reductase (EC 1.10.2.2), cytochrome oxidase (EC 1.9.3.1) and ATPase (EC 3.6.1.3)) in the mitochondria of liver, heart and brain of rats fed on diets differing extensively in their polyunsaturated fatty acid compositions have been investigated. The results showed that fatty acid compositional changes brought about by the dietary differences were associated with extensive changes in the activities of these key enzymes in the mitochondria. The extent of the influence differed considerably with the period over which the diets were fed. The role of dietary lipids to effect changes through the preservation of membrane structural integrity is discussed.

Adenosine Triphosphatases↗