C. Castelluccio

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.

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

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.

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.

Modes of coenzyme Q function in electron transfer

SummaryIn the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.

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.

Role of Ubiquinone Diffusion in Mitochondrial Electron Transfer

It is presently recognized that the transfer of electrons from substrates to molecular oxygen in mitochondria involves integral membrane protein complexes containing redox groups of increasing redox potentials (Tzagoloff, 1982). According to the original intuition of Green (1966), it is widely accepted that electron transfer between complexes is ensured by mobile components acting as cosubstrates, vz. ubiquinone (Q) and cytochrome c (cyt.c). In particular, ubiquinone holds a central position in the chain, being reduced by a large collection of flavoprotein dehydrogenases and reoxidized by the ubiquinol-cyt.c reductase complex (bql complex or Complex III)(Lenaz et al., 1985 b): such position is likely to mark a strategic point for a regulatory role in electron transfer.

Quality assurance in United Kingdom higher education. A case study: the London Metropolitan University

The aim of this study is to present the Quality Assurance System of one of the largest universities in Great Britain, the London Metropolitan University (London Met). A brief history of the evaluation system in the United Kingdom (UK) and a description of the current situation of Higher Education in the UK, as to the quality and standard assurance will be presented to start with. In particular, we will describe the activities carried out by the national Quality Assurance Agency and the quality procedures implemented in England, Northern Ireland, Scotland and Wales. As to the London Met, after a short presentation of the University, we will try to present an overview of the quality assurance and management systems, their facilities, the people involved (boards, committees, departments, internal and external examiners) and the procedures currently in place. Finally, we will describe the policy adopted by the London Met with respect to external accrediting agencies and professional bodies that also reported to the organisation of the QAA Institutional Audit in spring 2005.

Coenzyme Q changes in liver and plasma in the rat after partial hepatectomy

The levels of coenzyme Q were determined 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, whereas it was significantly lower in hepatectomized with respect to non-operated animals. Plasma CoQ9 levels decreased dramatically in hepatectomized animals, but increased strongly in sham-operated in comparison with non-operated rats. The data suggest the possibility of a rate-limiting step in CoQ biosynthesis in hepatectomized animals.