The action of digitonin on rat liver mitochondria. The effects on enzyme content

Abstract

1. The effects of repetitive treatment of rat liver mitochondria with digitonin were examined. The first treatment results in the removal of the outer membrane. Almost all the NADH–cytochrome c reductase (rotenone-insensitive) is lost whereas the major portions of the soluble and bound enzymes are retained. One exception appears to be the cytochromes, which undergo somewhat larger losses. The resulting inner-membrane complex carries out oxidative phosphorylation and Pi–ATP exchange. 2. The properties of the inner-membrane complex are affected by the osmoticity of the medium. When it is suspended in water little protein is lost but there is a marked loss of phosphorylation. If after the suspension in water the particulate fraction is reisolated by centrifugation and treated with digitonin, or if the aqueous suspension is treated directly with digitonin and the particulate fraction then reisolated, the phosphorylation is largely restored. 3. Additional treatment of the inner mitochondrial complex with digitonin results in the formation of a particulate fraction that contains approx. 8% of the initial mitochondrial protein, no outer membrane, no soluble mitochondrial enzymes and is still capable of coupled oxidative phosphorylation and Pi–ATP exchange. These effects cannot be reproduced by treatment with water. 4. The rat liver mitochondria and all of the resulting preparations obtained after digitonin treatment may be stored for long periods in dimethyl sulphoxide with little change of activity.