digitonin and carboxyatractyloside

The localization of the mitochondrial elongation activities ('elongases') from Saccharomyces cerevisiae has been investigated. It was shown, using carboxyatractyloside in the incubation mixture, that synthesis of very-long-chain fatty acids probably occurred outside the matrix and, by fractionation experiments, that elongases are membrane-bound enzymes. The solubilization of the outer membrane by digitonin showed that three elongating activities are correlated with a marker of the outer membrane and not with an inner membrane marker. A further partial purification of the outer membrane showed that elongases are present in the outer membrane of mitochondria.

Topics: Acyl-Carrier Protein S-Malonyltransferase; Acyltransferases; Atractyloside; Binding Sites; Centrifugation, Density Gradient; Digitonin; Enzyme Activation; Fatty Acids; Intracellular Membranes; Mitochondria; Oxygen Consumption; Saccharomyces cerevisiae; Sucrose

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Animals; Atractyloside; Cattle; Cytosol; Digitonin; Epididymis; Hexokinase; Male; Mitochondria; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Spermatozoa; Succinates