Hana Rauchová

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.

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 cytochromec 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 cytochromec 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 cytochromec 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 theKm for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. 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.

The effect of lysophosphatidylcholine on the activity of various mitochondrial enzymes

The influence of lysophosphatidylcholine (LPC) on H(+)-ATPase, cytochrome oxidase (COX), glycerolphosphate dehydrogenase (GPDH) and malate dehydrogenase (MDH) was followed. The activities of H(+)-ATPase and COX increased with increasing LPC concentration up to 0.5 mg/mg protein when maxima were achieved. This activatory effect is LPC-specific, because Lubrol-treated or frozen-thawed mitochondria showed lower activities of these enzymes. H(+)-ATPase was not influenced by higher concentration of LPC, while COX activity decreased with increasing amount of LPC. The activity of GPDH decreased at very low concentration of LPC and was not further modified at higher LPC concentration. In an attempt to find the concentration of LPC necessary for a complete permeabilization of inner mitochondrial membrane we followed the influence of lysolipid on the release of MDH activity from the mitochondrial matrix. The full activity of this enzyme was obtained with a concentration 0.75 mg LPC/mg protein indicating that mitochondria were completely broken. Our data indicate that LPC significantly affects activity of enzymes connected with mitochondrial membrane and can be useful for evaluation of the importance of phospholipid microenvironment for the enzyme function.

Regulation of glycerol 3-phosphate oxidation in mitochondria by changes in membrane microviscosity

The inhibition of glycerol 3‐phosphate oxidation by oleic acid correlates with changes in membrane microviscosity monitored by the steady‐state fluorescence anisotropy of DPH. The dynamic measurements indicate that the changes of both the limiting anisotropy and rotational relaxation time occur in a concentration range where the enzyme activity is strongly inhibited.

Influence of thyroid status on the membranes of rat liver mitochondria Unique localization of L-glycerol-3-phosphate dehydrogenase

The effect of thyroid status on the physical properties of rat liver mitochondrial membranes and on the lipid microenvironment of proteins was investigated. The steady‐state fluorescence anisotropy of diphenyl‐1,3,5‐triene and 1‐[4‐{trimethylaminophenyl} phenyl]‐6‐phenylhexa‐1,3,5‐triene revealed an increase of the order of the membranes with the increase of hormone level. Protein arrangement in the inner mitochondrial membrane altered with the thyroid status, which was reflected by digitonin subfractionation of mitochondria. The microenvironment of FAD‐linked L‐glycerol‐3‐phosphate dehydrogenase was dramatically influenced by thyroxine.

Phosphoenolpyruvate shuttle - transport of energy from mitochondria to cytosol

Brown‐adipose tissue mitochondria of hamster and rat contain phosphoenolpyruvate carboxykinase (EC 4.1.1.32). In the presence of ketoglutarate and malate, phosphoenolpyruvate is formed and exported forms mitochondria. Phosphoenolpyruvate formation is inhibited by 1,2,3‐benzenetricarboxylate. It is proposed that phosphoenolpyruvate carboxykinase together with pyruvate carboxylase and pyruvate kinase forms a phosphoenolpyruvate shuttle through which energy produced by the Krebs cycle in mitochondria may be exported to cytosol.

Inhibition of mitochondrial ATPase by dicarbopolyborate, a new enzyme inhibitor

Polyborate anions were found to inhibit mitochondrial ATPase. Mercapto and chloro derivatives of dicarbononaborates showed full inhibition of the enzyme activity at 0.5–0.8 mM. The inhibitory effect of dodecaborates was lower. The inhibition was of competitive type with respect to ATP. The inhibition of soluble F1-ATPase indicates a direct interaction of the polyborate anion with the catalytic part of the enzyme molecule.

Oxidative metabolism of the inner and outer ventricular layers of carp heart (Cyprinus carpio L.)

Abstract 1. 1. The biochemical characteristics of homogenates and mitochondria isolated from the outer and inner layers of the ventricular myocardium of carp were studied. 2. 2. The homogenate prepared from the inner layer exhibited higher activity of cytochrome oxidase than that from the outer layer. No difference was found in the activity of cytochrome oxidase between mitochondria from the inner and outer layers. Difference spectra of cytochromes also showed that their content in mitochondria of both layers is similar and that the higher oxidative capacity of the spongious layer is due to a higher content of mitochondria. 3. 3. In comparison with rat heart a higher content of cyt aa3 and a lower content of Cyt b and cyt cc1 were found in carp heart mitochondria. 4. 4. In comparison with rat heart, carp heart mitochondrial enzymes were more sensitive to freezing-thawing and to detergent action.

Incorporation of mitochondrial l-glycerol-3-phosphate dehydrogenase into liposomes; effect of sodium oleate and calcium ions

FAD-linked L-glycerol-3-phosphate dehydrogenase purified from liver mitochondria of hyperthyroid rats was incorporated into unilamellar phospholipid liposomes. The incorporation influenced both Vmax,app and Km,app values of the enzyme for its substrate, L-glycerol 3-phosphate. The Km,app for the electron acceptor remained unchanged with a simultaneous slight enhancement of the corresponding Vmax,app value. The steady-state fluorescence anisotropies of the fluorescein isothiocyanate and trimethylammoniumdiphenylhexatriene labels were affected by sodium oleate and calcium ions in the case of both solubilized and liposome-incorporated L-glycerol-3-phosphate dehydrogenase. These results indicate that calcium ions cause a significant alteration of the enzyme conformation. Sodium oleate (used as a model of free fatty acids), besides its direct action on the enzyme itself, affects the enzyme indirectly as well, via alteration of the physical properties of the membrane.

Inhibition of the Mitochondrial ATPase by Polyborate Anions Designed for Neutron Capture Therapy

Mercapto undecahydro dodecaboron designed for neutron capture therapy and several new carboranes showed inhibitory effects on mitochondrial ATPase activity. For mercapto and chloroderivates of carboranes 50% inhibition of ATPase activity was found at 100–200 uM concentration. For dodecaboranes 50% inhibition was found at 10-times higher concentration.