ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1.

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different

Evaluation By Blue Native Polyacrylamide Electrophoresis Colorimetric Staining Of The Effects Of Physical Exercise On The Activities Of Mitochondrial Complexes In Rat Muscle.

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 +/- 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 +/- 0.65, P < 0.05) and soleus (9.8 +/- 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.37939-4

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.93994

Kinetic studies with competitive inhibitors indicate a sequential mechanism for dopamine beta-hydroxylase.

Competitive inhibitors have been used to study the kinetic mechanism of action of the multireactant enzyme dopamine beta-hydroxylase (dopamine beta-monooxygenase EC 1.14.17.1). The copper chelator penicillamine, which was found to be a competitive inhibitor of ascorbate, gave noncompetitive inhibition with respect to tyramine. The antidopaminergic drug cis-chlorprothixene, a competitive inhibitor for tyramine, produced uncompetitive inhibition with respect to ascorbate. These findings are consistent with an ordered sequential mechanism, with ascorbate as the initial substrate to add to the enzyme. The pharmacological relevance of dopamine beta-hydroxylase inhibition by penicillamine is also discussed

Structural and functional modifications induced by diamide on the F0 sector of the mammalian ATP synthase

none3In this report data are presented which firmly establish that by treating isolated F0 with the thiol reagent diamide, two 25 kDa F0 subunits react to form a dimer of 45 kDa apparent molecular mass. This dimerising effect is correlated to the impairment of the binding of F1 to F0, both at microM and mM diamide concentrations. Under the latter condition, modification of other F0 subunits also occurs. Passive proton conductance through F0, as well as its sensitivity to N,N'-dicyclohexylcarbodiimide, are affected at low diamide concentration. Thus perturbation of the cysteine residue of the 25 kDa F0 subunit is sufficient for altering the ATP synthase proton channel.noneDABBENI-SALA F.; LIPPE G.; M. SORGATODABBENI SALA, F.; Lippe, G.; Sorgato, MARIA CATI

Molecular regulation of cholesterol metabolism: HDL-based intervention through drugs and diet.

The overloading of cholesterol in the arteries remains the principal cause of cardiovascular diseases. Since available anticholesterolemic drugs are not completely effective and have several severe adverse effects, the aim of this review is to analyze current research focused on the emerging, innovative therapeutic strategies based on both pharmacological and nutritional interventions to control cholesterol metabolism. Pharmacological interventions mainly involve the use of molecules capable of interfering with high-density lipoprotien (HDL) metabolism and the reverse cholesterol transport (RCT) through genetic control of apolipoprotein A-I (ApoA-I), agonism at liver X-receptor alpha (LXRalpha), or inhibition of cholesteryl ester transport protein (CETP), scavenger receptor BI(SR-BI), and ecto F0F1ATPase/synthase. Nutritional interventions are based on the use of fibres, phytosterols, and probiotics acting through interference with absorption and reabsorption of cholesterol by enterocyte and hepatocyte specific transporters, thus influencing RCT final step. The search for new drugs is still at the very beginning and new molecules are not yet ready to enter clinical use. However, several promising findings coming from innovative biotechnological research are expected shortly to produce probiotics, fibres, and phytosterols to be used as therapeutic tools. Among the most important advantages of natural products in respect to traditional drugs are the lack of severe adverse effects and their low cost

Structural and functional modifications induced by diamide on the F0 sector of the mammalian ATP synthase

In this report data are presented which firmly establish that by treating isolated F0 with the thiol reagent diamide, two 25 kDa F0 subunits react to form a dimer of 45 kDa apparent molecular mass. This dimerising effect is correlated to the impairment of the binding of F1 to F0, both at microM and mM diamide concentrations. Under the latter condition, modification of other F0 subunits also occurs. Passive proton conductance through F0, as well as its sensitivity to N,N'-dicyclohexylcarbodiimide, are affected at low diamide concentration. Thus perturbation of the cysteine residue of the 25 kDa F0 subunit is sufficient for altering the ATP synthase proton channel