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Intact Mitochondria (intact + mitochondria)
Selected AbstractsMitochondrial affinity for ADP is twofold lower in creatine kinase knock-out musclesFEBS JOURNAL, Issue 4 2005Possible role in rescuing cellular energy homeostasis Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK,/,), mitochondrial (Mi-CK,/,) and double knock-out (MiM-CK,/,), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2·mg mitochondrial protein,1·min,1) and ADP affinity (; µm) were determined by respirometry. State 3 Vmax and of M-CK,/, and MiM-CK,/, gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK,/,. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK,/, phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK,/, muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes. [source] Submitochondrial localization of 6- N -trimethyllysine dioxygenase , implications for carnitine biosynthesisFEBS JOURNAL, Issue 22 2007Naomi Van Vlies The first enzyme of carnitine biosynthesis is the mitochondrial 6- N -trimethyllysine dioxygenase, which converts 6- N -trimethyllysine to 3-hydroxy-6- N -trimethyllysine. Using progressive membrane solubilization with digitonin and protease protection experiments, we show that this enzyme is localized in the mitochondrial matrix. Latency experiments with intact mitochondria showed that 3-hydroxy-6- N -trimethyllysine formation is limited by 6- N -trimethyllysine transport across the mitochondrial inner membrane. Because the subsequent carnitine biosynthesis enzymes are cytosolic, after production, 3-hydroxy-6- N -trimethyllysine must be transported out of the mitochondria by a putative mitochondrial 6- N -trimethyllysine/3-hydroxy-6- N -trimethyllysine transporter system. This transport system represents an additional step in carnitine biosynthesis that could have considerable implications for the regulation of carnitine biosynthesis. [source] Metabolic effects of carbenoxolone in rat liverJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2006Leandro Silva Pivato The action of carbenoxolone on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In perfused livers, carbenoxolone (200,300 ,M) increased oxygen consumption, glucose production and glycolysis from endogenous glycogen. Gluconeogenesis from lactate or fructose, an energy-dependent process, was inhibited. This effect was already evident at a concentration of 25 ,M. The cellular ATP levels and the adenine nucleotide content were decreased by carbenoxolone, whereas the AMP levels were increased. In isolated mitochondria, carbenoxolone stimulated state IV respiration and decreased the respiratory coefficient with the substrates ,-hydroxybutyrate and succinate. The ATPase of intact mitochondria was stimulated, the ATPase of uncoupled mitochondria was inhibited, and the ATPase of disrupted mitochondria was not altered by carbenoxolone. These results indicate that carbenoxolone acts as an uncoupler of oxidative phosphorylation and, possibly, as an inhibitor of the ATP/ADP exchange system. The inhibitory action of carbenoxolone on mitochondrial energy metabolism could be contributing to induce the mitochondrial permeability transition (MPT), a key phenomenon in apoptosis. The results of the present study can explain, partly at least, the in vivo hepatotoxic actions of carbenoxolone that were found in a previous clinical evaluation. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:230,240, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20139 [source] Metabolic effects of p -coumaric acid in the perfused rat liverJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2006Leonardo C.N. Lima The p -coumaric acid, a phenolic acid, occurs in several plant species and, consequently, in many foods and beverages of vegetable origin. Its antioxidant activity is well documented, but there is also a single report about an inhibitory action on the monocarboxylate carrier, which operates in the plasma and mitochondrial membranes. The latter observation suggests that p -coumaric acid could be able to inhibit gluconeogenesis and related parameters. The present investigation was planned to test this hypothesis in the isolated and hemoglobin-free perfused rat liver. Transformation of lactate and alanine into glucose (gluconeogenesis) in the liver was inhibited by p -coumaric acid (IC50 values of 92.5 and 75.6 ,M, respectively). Transformation of fructose into glucose was inhibited to a considerably lower degree (maximally 28%). The oxygen uptake increase accompanying gluconeogenesis from lactate was also inhibited. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC50 = 160.1 ,M); no such effect was observed in freeze,thawing disrupted mitochondria. Glucose 6-phosphatase and fructose 1,6-bisphosphatase were not inhibited. In isolated intact mitochondria, p -coumaric acid inhibited respiration dependent on pyruvate oxidation but was ineffective on respiration driven by succinate and ,-hydroxybutyrate. It can be concluded that inhibition of pyruvate transport into the mitochondria is the most prominent primary effect of p -coumaric acid and also the main cause for gluconeogenesis inhibition. The existence of additional actions of p -coumaric acid, such as enzyme inhibitions and interference with regulatory mechanisms, cannot be excluded. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:18,26, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20114 [source] A critical comparison between two classical and a kit-based method for mitochondria isolationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2009Sonja Hartwig Abstract Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method. Our analyses show that the sophisticated (and most expensive) free-flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time-consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations. [source] | ||||||||||