The binding of cationic dyes to energized submitochondrial particles and the extent and rate of absorbance changes accompanying the binding are enhanced by the presence of inorganic anions. The anion effect follows the sequence : SCN-> C10,-> I-> NO,-> Br-> C1-> Br03-= H2P0,-> CH,COO-> F--. With the more lipophilic anions the phase of enhancement is followed by one of inhibition: this effect is more marked with more lipophilic dyes. The rate and extent of absorbance changes is also affected by cations according to the sequence:Under conditions where the anions markedly enhance the binding of the dyes by the energized fragments there is no parallel increase of anion binding. It is suggested that the membrane conformation and therefore the accessibility of the sites for various ligands, is modified during the interaction of electrolytes with energized fragments.Several reactions in submitochondrial particles are markedly affected by the electrolyte composition of the medium [1][2][3][4][5][6][7][8]. One interpretation for these effects, assumes formation of a membrane potential, diffusion of the anions across the membrane down the electrical gradient, collapse of the potential and anion accumulation [3,6,9-111. This mechanism is considered to be supported by the energy-linked accumulation of I -anilinonaphthalene 8-sidphonic acid, phenyl dicarbaundecarborane anion and SCN- The alternative interpretation, on the other hand, assumes that the mitochondria1 membrane contains energy-linked rearranging chemical groups, which are capable of interaction with ligands solubilized in microenvironments of the membrane fabric [ 14-16]. Acridines and aminophenazine dyes behave as ligands interacting with specific membrane sites [15]. We now report a number of observations suggesting that the r6le of the anions, on the energy-linked responses of cationic probes, is not primarily related to the process of energy conservation but rather to the structural requirements which determine the accessibility of each probe for the membrane site. We propose that the dependence of the anion effects on the lyotropic series be better interpreted in terms of the basic Hofmeister concept [l 71 originating from experiments on protein conformation.
MATERIALS AND METHODSPreparations of rat liver mitochondria were made according t o standard procedures. The preparation of beef heart mitochondria has been described previously [ 151. The sonication of mitochondria was carried out either in the presence of 2 mM EDTA pH 8.5, or of 10mM MgCl,, 1 mM ATP, 2 mM succinate, 10mM Tris pH8.5 or of 20mM pyrophosphate. Essentially similar results were obtained with either the EDTA or the Mgz+ or the pyrophosphate particles. The binding of the cationic dyes and the absorbance changes of the dyes following the energization of the fragments in the presence of the various incubation media was carried out as described previously [15]. I n the case of the spectrophotometric experiments, two parameters were followed. The first was the extent of absorbance change; after ...