The effect of butylcaptax on oxidative phosphorylation and the activity of multienzyme systems of the mitochondrial membranes in the rat liver]

The oxidative phosphorylation and the activity of polyenzymatic systems in the rat liver mitochondrial membranes were studied upon a 5 day administration to rats of butylcaptax in the dose of 1/10 LD50. It is established that butylcaptax led to significant disturbances in the system of oxidative phosphorylation and in the transfer of electrons in the rat liver mitochondrial respiratory chain.     

Various properties of ribonucleotide reductase from rat liver mitochondria]

Three fractions of mitochondria (light, heavy and intermediate) from normal and regenerating rat liver ( in 15, 24 and 48 hours after partial hepatectomy) were isolated by means of differential centrifugation. Heavy mitochondrial fraction was shown to have a higher CDP-reductase activity than light mitochondria. The activity of the procces of ribonucleotides reduction in the mitochondria was shown to depend on the tissue functional state and it was maximal in 24 hours after partial hepatectomy.     

Investigation of ribosomal RNAs from liver mitochondria of albino rats]

Study of sedimentation coefficients of rat liver mt-rRNAs labelled in vivo under conditions of selective inhibition by actinomycin D of nuclear RNAs biosynthesis, in hypoosmic system of isolated mitochondria, and of mt-rRNAs isolated from purified 55 ribosomes revealed that mt-rRNAs are represented by 16S and 12S components. Both components are found to be of AU-type. Anomalous behaviour of mtRNA on MAK columns is observed. Some aspects of isolation and identification of mt-rRNAs in higher organisms are discussed.     

The interaction of lipoic acid and its metabolites with aspartate and alanine aminotransferases in a multienzyme complex from rat liver and brain mitochondria]

It is shown that lipoic acid and its metabolites--lipoamide and 4,6-dithiohexanoic acid can interact and inhibit the mitochondrial aspartate- and alanine-aminotransferase activities. This effect is much more pronounced in the case of multienzyme complexes--metabolon.     

Some permeability properties of isolated rat liver cell mitochondria

The rates of penetration of various solutes into isolated rat liver mitochondria have been studied. Sodium, potassium, and sucrose were observed to enter the mitochondria until an equilibrium concentration was reached. The diffusion of these solutes, after the first few minutes, followed the predicted diffusion curve for solutes entering a particle with a rate-limiting membrane and instantaneous mixing in the interior. Reasons for deviations from the predicted equation during the first few minutes of diffusion are suggested. The data show that at pH 7.4 sodium and potassium enter more rapidly than sucrose. I¹³¹-labelled albumin was found to enter very slowly, if at all. Increasing the pH from 7.4 reduced the rate at which sodium ion penetrated the mitochondria. The rate of diffusion of sucrose into mitochondria was considerably slower than diffusion of sucrose into a sphere of water of the same size. Sodium ion was not found to be concentrated in vitro against an external concentration gradient as has been reported by other investigators. It is concluded that the rate of diffusion of solutes between the external medium and the interior of mitochondria is probably restricted and controlled by a mitochondrial membrane exhibiting passive permeability characteristics.     

Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver

The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.     

Quantitative characteristics of glutamate transport in rat liver mitochondria

1. The kinetics of glutamate transport into mitochondria were determined by using Bromocresol Purple to terminate the transport process. 2. Glutamate transport was found to have a V(max.) of 9.1nmol/min per mg of protein at pH6.9 and 20 degrees C; the K(m) for glutamate was 4mm. 3. The rate of glutamate deamination in intact mitochondria was tenfold slower than in disrupted mitochondria. 4. These results suggest that glutamate deamination may be controlled by the rate of glutamate transport. Possible consequences of these findings are discussed.     

Resistance to heating and trypsin of glutamate dehydrogenase from liver mitochondria of frogs differing in thermotaxis]

The resistances to heat and trypsin hydrolysis served as indirect indices of conformational flexibility of glutamate dehydrogenase molecules. No difference in heat resistance was found between crystalline eletrophoretically homogeneous preparations of glutamate dehydrogenase from liver mitochondria of two species of frogs, the more southern Rana ridibunda and the more northern R. temporaria. However, glutamate dehydrogenase from R. ridibunda is digested by trypsin at a lower rate than that from R. temporaria, which may be explained by its lower conformational flexibility. Therefore positive correlation between conformational flexibility of glutamate dehydrogenase and mean ambient temperature of the species studied is revealed only with respect to resistance to proteolysis.     

Inhibition of mitochondrial function in isolated rat liver mitochondria by azole antifungals

Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related drug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I₅₀ was 32, 300, and 110 μM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I₅₀ of 350 μM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I₅₀ of 74 μM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action. © 1997 John Wiley & Sons, Inc.