Close location of the first loop to the third loop of the mitochondrial ADP/ATP carrier deduced from cross-linking catalyzed by copper-o-phenanthroline of the solubilized carrier with Triton X-100.
Effects of the cross-linking catalyst copper-o-phenanthroline [Cu(OP)2] on the bovine heart mitochondrial ADP/ATP carrier solubilized with Triton X-100 were studied under various conditions. Without detergent treatment, Cu(OP)2 specifically catalyzed the formation of intermolecular disulfide bridges in submitochondrial particles between two Cys56 residues in the first loop facing the matrix space of the dimeric carrier [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J. Biol. Chem. 270, 29548-29554]. However, an intramolecular disulfide bridge between Cys56 and Cys256 in the third loop was formed in the solubilized carrier. Proteolytic digestion of the carrier with lysylendopeptidase showed that it first cleaves the Lys42-Gln43 bond and then the Lys48-Gln49 bond of the first loop in the membrane-bound carrier, but it cleaves both sites almost simultaneously in the solubilized carrier. These features were observed only with the m-state carrier; the c-state carrier was not subject to any cross-linking or proteolytic digestion. It is suggested that the protruding first loop is located close to the third loop, which could be exposed to a certain degree.
Majima
Eiji
E
Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima 770-8505, Japan.
Takeda
Masashi
M
Miki
Satomi
S
Shinohara
Yasuo
Y
Terada
Hiroshi
H
D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives* / Q000494:pharmacology; D001865:Bongkrekic Acid / Q000494:pharmacology; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D017830:Octoxynol / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000378:metabolism; D010618:Phenanthrolines / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012995:Solubility; D013194:Staining and Labeling
D003432:Cross-Linking Reagents; D004220:Disulfides; D010618:Phenanthrolines; D001865:Bongkrekic Acid; D001278:Atractyloside; C033531:eosin maleimide; D017830:Octoxynol; D000226:Mitochondrial ADP, ATP Translocases; D010447:Peptide Hydrolases; C003853:carboxyatractyloside; D004801:Eosine Yellowish-(YS); C025205:1,10-phenanthroline