Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides.
The effects of six thiol-specific cross-linker dimaleimides, in which the distance of the two maleimide groups ranged from 7.7 to 16. 8 A, on bovine heart mitochondria were studied at pH 7.2 and 0 degrees C. None of the dimaleimides affected mitochondrial proteins, but they caused significantly specific intermolecular cross-linking of the 30 kDa ADP/ATP carrier in submitochondrial particles. All the cross-links were found to be formed specifically between two Cys56 residues in the first loop facing the matrix, as we observed previously in intermolecular disulfide bridge formation catalyzed by copper o-phenanthroline [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J.Biol. Chem. 270, 29548-29554]. The dimerization was dependent on the cross-linking span of the dimaleimides, being maximum with the dimaleimide having a span of about 12 A. Cross-linking took place in the m-state carrier, but not in the c-state carrier, and inhibited ADP transport via the ADP/ATP carrier. We suggest that a pair of first loops with Cys56 residues in the dimer form of the m-state carrier fluctuates widely with a most probable distance between them of about 12 A, and that this fluctuation modulates the transport activity of the ADP/ATP carrier.
Hashimoto
M
M
Faculty of Pharmaceutical Sciences, University of Tokushima, Japan.
Majima
E
E
Goto
S
S
Shinohara
Y
Y
Terada
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D005033:Ethylmaleimide / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry*; D011487:Protein Conformation; D012996:Solutions; D013367:Submitochondrial Particles / Q000737:chemistry
D003432:Cross-Linking Reagents; D008301:Maleimides; D012996:Solutions; C075594:1,6-bismaleimidohexane; D000244:Adenosine Diphosphate; D000226:Mitochondrial ADP, ATP Translocases; C011328:N,N'-4-phenylenedimaleimide; D005978:Glutathione; D003545:Cysteine; D005033:Ethylmaleimide; C023860:N,N'-2-phenylenedimaleimide