The identification of the site of action of NN'-dicyclohexylcarbodi-imide as a proteolipid in mitochondrial membranes.

Mitochondrial membranes were incubated with NN'-dicyclohexyl[(14)C]carbodi-imide, which irreversibly inhibited the partial reactions of oxidative phosphorylation by 95-100%. Solutions of the membranes were analysed on polyacrylamide gels. Of the radioactivity recovered from the gels 90% was shown to be associated with a single protein of molecular weight about 10000. The radioactive protein and associated phospholipid was solubilized from the membrane by extraction with chloroform-methanol mixtures and was concentrated 50-fold by solvent fractionation and adsorption chromatography on Sephadex LH-20. Several protein-radioactivity peaks were obtained by Sephadex LH-20 chromatography. However, 90-100% of the radioactivity in each peak was shown to be associated with a single protein similar to the major radioactive protein observed in electrophoretograms of the membrane solutions. It is concluded that dicyclohexylcarbodi-imide inhibits mitochondrial oxidative phosphorylation by reacting covalently with a group on this chloroform-methanol-soluble protein. The possible role of this protein in oxidative phosphorylation is discussed.