Studies of the Energy‐Transfer System of Submitochondrial Particles

Abstract

Submitochondrial particles elicit a fluorescence of 8‐anilino‐naphthalene‐1‐sulfonate which is increased when the particles are brought into the energized state. Various parameters of the anilino‐naphthalene sulfonate response were investigated in “EDTA particles” from beef‐heart mitochondria in the energized and non‐energized states. The results reveal striking differences between teh two types of anilino‐naphthalene sulfonate response in several respects, including the effects of pH and ionic strength, the number of binding sites, the dissociation constant, and the quantum yield. Conditions are established which result in a maximal ratio of energy‐dependent to non‐energy‐dependent anilino‐naphthalene sulfonate response and a virtually linear relationship of both types of response to particle concentration. It is shown that, under such conditions, there is a linear inverse relationship between the steady state of energy‐dependnet anilino‐naphthalene sulfonate response and the respiratory rate of the particles as measured with succinate as substrate in the presence of oligomycin and increasing concentrations of an uncoupler. Extrapolation of this straight line to respiratory rate = 0 allows an estimation of the extent of anilinonaphthalene sulfonate response at complete respiratory control, i.e., in the maximally energized state. Experiments with different substrates and electron‐transport inhibitors reveal that the linear relationship between the extent of energy‐dependent anilino‐naphthalene sulfonate response and the respiratory rate in the presence of increasing concentrations of uncoupler holds only when the cytochrome b‐c1 step of the respiratory chain is rate‐limiting. The available evidence indicates that the energy‐dependent anilino‐naphthalene sulfonate response originates mainly or exclusively from a restricted region or locus of the membrane, which is closely related to energy‐coupling site II of the respiratory chain. With ATP as the source of energy, the anilino‐naphthalene sulfonate response increases with low, and decreases with high, concentrations of oligomycin, in a fashion similar to that earlier found for oxidative phosphorylation and its reversal.