Oxidative phosphorylation in rat liver mitochondria is uncoupled by y-(p-arsenophenyl) -nbutyrate. The uncoupling activity is enhanced by several monothiol compounds but reversed by 2,3-dimercaptopropanol. The arsenical also stimulates the latent ATPase activity, and the stimulation is greater in the presence of 2-mercaptoethanol, 2-mercaptoethylamine, and thiophenol but unchanged by thioglycollate, cysteine, and glutathione. In the presence of a high concentration of arsenophenylbutyrate, the monothiols have only a small and varied effect but 2,3-dimercaptoethanol effectively reverses the ATPase stimulation. The data provide further support for the postulated participation of a dithiol group in oxidative phosphorylation. Arsenophenylbutyrate restores respiration which has been inhibited by oligomycin in initially tightly coupled mitochondria. Furthermore, oligomycin inhibits the ATPase evoked by the arsenical. Similar results have been obtained with cadmium ion, and with arsenite in combination with 2,3-dimercaptopropanol. It is concluded that the postulated dithiol site must be localized between the electron transport chain and the oligomycin-sensitive terminal coupling reaction.Recent work from this laboratory established that cadmium ion (Cd++) and arsenite in the presence of 2,3-dimercaptopropanol uncouple oxidative phosphorylation and stimulate the latent ATPase activity of mitochondria (Jacobs et al., 1956; Sanadi, 1960, 1961; Fletcher and %midi, 1962;; Fluharty and S a~d i , 1962b). The effects appeared closely parallel to those of 2,4-dinitrophenol. Arsenite and C d + + were considered to produce these effects by binding a dithiol function (Le., two juxtaposed --SH groups), although the requirement for dimercaptopropanol to potentiate the effects of arsenite had no previous analogy. The interpretation with respect to C d + + is also open to some criticism on the basis of its possible binding by nucleotide phosphate groups and chelation with nitrogen and sulfur (Vallee et al., 1961). However, since then, myosin ATPase (Fluharty and Sanadi, 1962a) and acetyl CoA carboxylase (Hatch and Stumpf, 1961) have beexi observed to exhibit a similar dithiol requirement to evoke the sensitivity to arsenite. In order to confirm the interpretation regarding dithiol involvement, we present in this paper data on the uncoupling and ATPase-stimulating effects of y-(p-arsenopheny1)-nbutyrate, a reagent whose specScity to dithiol groupings has been established by R e i (1958). The site of action of such dithiol-specifk reagents in the oxidative phosphorylation reactions has been localized to some extent by the use of oligomycin, a reagent introduced by Lardy and co-workers (1958).
EXPERIMENTAL PROCEDUREThe preparation of rat liver mitochondria and the assays for oxidative phosphorylation and ATPase activity have been described in earlier communications (Jacobs et al., 1956; Sanadi, 1960,1961).In these experiments the uncoupling agent and thiol compounds were incubated routinely for 10 minutes in a n ice bath with the mitoch...