G. Parenti Castelli

Mitochondrial bioenergetics in aging.

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.

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 ‘K m’ for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The K m 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.

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.

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.

The function of coenzyme Q in mitochondria

SummaryWe 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

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.

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].

Effects of cholesterol on the kinetics of mitochondrial ATPase.

Enrichment of the inner mitochondrial membrane with cholesterol induces an increase in ATPase activity with a decrease in the K m for ATP. Cholesterol also abolishes the discontinuity normally found in the Arrhenius plot of ATPase activity. Since no change is detected in the rate of proton translocation through the ATPase membrane sector, it is concluded that cholesterol incorporation induces changes in the hydrolytic step of ATPase via a conformational change transmitted from the membrane sector to the catalytic sector F1.

The kinetic and structural changes of the mitochondrial F1-ATPase with temperature

Mitochondrial F1-ATPase shows a break in the Arrhenius plot with an increase of the activation energy below 17 degrees C, this may imply that the F1-ATPase undergoes a conformational change at this temperature. Further, a structural change of the F1-ATPase is indicated by analysis of the intrinsic fluorescence at 307 nm between 33 and 11 degrees C and also by evaluation of the circular dichroism spectra of the enzyme at temperatures below and above the temperature corresponding to the discontinuity of the Arrhenius plot. It is therefore suggested that F1-ATPase exists in two temperature dependent conformational states to which different catalytic properties may be assigned.

Nonionic interaction between proteins and lipids in the mitochondrial membranes

Abstract The interaction between lipid-depleted mitochondria and phospholipids is not inhibited by high ionic strength of the medium, and therefore is not electrostatic but is conceivably hydrophobic in nature. The proteins responsible for the interaction, both in high and low ionic strength media, are located in the basepieces of the membrane. These findings are not compatible with the unit membrane model of a lipid bilayer sandwiched between two extended protein layers.