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

Abstract

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.

The proton conducting F0-part of bacterial ATP synthases

“…The binding of [14C]DCCD to subunit c (proteolipid) in all analyzed organisms was rather speeifie and oeeurred at low eoneentration (1-10 nmoljmg membrane protein) [6,118]. Maximum inhibition was observed when only a fraction of the subunits was labelIed, indicating a cooperative interaetion of the subunits [26,27,[118][119][120][121][122][123].…”

“…Maximum inhibition was observed when only a fraction of the subunits was labelIed, indicating a cooperative interaetion of the subunits [26,27,[118][119][120][121][122][123]. For the beef-heart ATP synthase, the existenee of a dimer of two proteolipids binding [14C]DCCD with a different affinity [123,124] compared with the monomerie subunits has been demonstrated.…”

Identification of the DCCD‐reactive protein of the energy transducing adenosinetriphosphatase complex from Escherichia coli

“…The vesicles were then sedimented by centrifuging at 40 000 g for 30 min and were washed three times with the same medium. The DCCD-reactive protein was extracted from the ['"Cl DCCD-labeled membranes by the method of Cattell et al [18].…”

“…It is the aim of this article to describe the identification of the DCCD-reactive protein from E.coli membranes by use of the radioactively labeled inhibitor. This approach, as already described for mitochondrial membranes [ 18,191 , is feasible since the carbodiimide is irreversibly bound to the site of action within the membrane.…”

Is the mitochondrial dicyclohexylcarbodiimide‐reactive protein of Mr 33 000 identical with the phosphate transport protein?

“…Dicyclohexylcarbodiimide acts at low concentrations as a specific inhibitor of H'-ATPase due to the covalent binding to a hydrophobic subunit of F, [1,2]. In mammalian mitochondria, however, an additional protein of different M, (33 000) is also labelled at H'-ATPase-inhibitor concentrations of ['4C]DCCD f2-41.…”