The fractionation of liver mitochondria with digitonin

• 1.1. Oxidative phosphorylation has been measured in the digitonin particle P₁ of Cooper and Lehninger [3] with glucose and hexokinase as a trap for the ATP produced. With β-hydroxybutyrate as substrate, the mean P:O ratio was 1.0 (highest 1.5) and the mean Q_O2 22 (highest 38), Phosphorylation of a low order (0.20) also has been found to be coupled to the oxidation of succinate. The pH-activity curve of the P:O ratio with β-hydroxybutyrate showed a narrow pH optimum at pH 6.5. The Q_O2 of the digitonin particle is 2.2 times that of normal mitochondria with β-hydroxybutyrate as the substrate.
• 2.2. The effect of sucrose on the fragmentation of rat-liver mitochondria with 1 per cent digitonin, as reflected by the protein distribution, P:O ratio and Q_O2, has been studied. From these studies it can be concluded that the digitonin has two effects, namely (1) it damages intact mitochondria by swelling and uncoupling prior to splitting; (2) it splits the mitochondria into smaller particles in such a way that a number of enzymes such as glutamic dehydrogenase are lost, but phosphorylation with β-hydroxybutyrate is not affected, and is independent of particle size. On the other hand, the phosphorylation with succinate is greatly decreased and is dependent on particle size. Both of these effects are inhibited by sucrose.
• 3.3. The same three DNP-stimulated ATP-ases found by Myers and Slater [10] in mitochondria also occur in the digitonin particle but are active in the absence of DNP. However, most of the 9.4 enzyme also present in intact mitochondria is not retained in the particles.