antimycin and alpha-glycerophosphoric-acid

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.

Topics: Animals; Antifungal Agents; Antimycin A; Brain; Cell Respiration; Electron Transport; Electron Transport Complex I; Electron Transport Complex III; Glycerolphosphate Dehydrogenase; Glycerophosphates; Guinea Pigs; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Methacrylates; Mitochondria; NADP; Reactive Oxygen Species; Subcellular Fractions; Succinic Acid; Thiazoles; Uncoupling Agents

The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes.

Topics: Aminooxyacetic Acid; Animals; Antimycin A; Aspartic Acid; Carcinoma, Ehrlich Tumor; Cytosol; Fructose; Glucose; Glycerophosphates; Glycolysis; Kinetics; Lactates; Malates; Mice; Mice, Inbred C57BL; NADH Dehydrogenase; Oxidation-Reduction; Rotenone