Oxidative phosphorylation is a vital process in the economy of aerobic cells, probably accounting for over 90 percent of all the adenosine triphosphate (ATP) generated from adenosine diphosphate (ADP) and phosphate at the expense of the energy liberated during catabolism of foodstuffs. Despite the great importance of this process and 20 years of intensive investigation, the enzymatic-details of oxidative phosphorylation are still essentially unknown. It is certain, however, that during the exergonic passage of a pair of electrons from substrate to molecular oxygen, via the pyridine nucleotides, flavoprotein, and the cytochromes, 3 moles of ATP are generated in coupled reactions occurring at three points in the respiratory chain (1-3), presumably those shown in the upper portion of Fig. 1.Both electron transport and oxidative phosphorylation take place in mitochondria, the structure of which must be relatively intact in order that oxidative phosphorylation may be observed in vitro. This finding has greatly limited the experimental approaches to the enzymatic mechanisms involved; it is largely for this reason that the mechanism of oxidative phosphorylation remains one of the most conspicuous mysteries in contemporary biochemistry.In the last three years we have studied experimental procedures for making morphologically less organized preparations from rat liver mitochondria with which the enzymatic details of oxidative phosphorylation could be more directly studied, with the ultimate goal of resolving the mechanism of oxidative phosphorylation by enzyme separation and reconstruction approaches. We have found it possible to obtain fragments of the mitochondrial membrane which contain relatively intact respiratory chain assemblies, including the enzymes neces-450 sary for coupling phosphorylation to electron transport. This article summarizes the chemical and enzymatic properties of these membrane fragments, prepared by the action of digitonin on rat liver mitochondria, and the new information they have yielded concerning the mechanism of oxidative phosphorylation and its relation to other aspects of mitochondrial physiology.
*Membrane FragmentsPreparation. Washed rat liver mitochondria are suspended in 0.05M sucrose and treated in the cold with an 0.8-percent solution of recrystallized, metal-free digitonin. The mitochondria undergo virtual dissolution, leaving a turbid brown solution which is centrifuged at 50,000 g for 25 minutes. The supernatant fluid, which is not clear but contains a gelatinous material, is then centrifuged at 100,000 g for 25 minutes to sediment the phosphorylating membrane fragments (4, 5). When suspensions of this preparation are incubated aerobically with D-(3-hydroxybutyrate, ADP, and orthophosphate, oxygen is taken up, acetoacetate accumulates in equivalent amounts, and inorganic prosphate (Pi) disappears with equivalent formation of ATP. The observed P:2e ratios (moles of Pi taken up per pair of electrons transferred) have been as high as 2.8 [maximum expected, 3.0 (2)] but are more usua...
