Irreversible extrusion of the first loop facing the matrix of the bovine heart mitochondrial ADP/ATP carrier by labeling the Cys(56) residue with the SH-reagent methyl methanethiosulfonate.
The effect of the SH-reagent methyl methanethiosulfonate (MMTS) on the ADP/ATP carrier of bovine heart mitochondria was studied under various conditions. MMTS labeled predominately Cys(56) in the first loop facing the matrix (loop M1), and the labeling inhibited ADP transport via the carrier. The transport inhibition was found to be due to fixation of the carrier in the m-state conformation. MMTS labeling was suggested not to affect ADP binding to its major binding site. These features were the same as those of another commonly used SH-reagent, N-ethylmaleimide (NEM). Although the van der Waals volume of the non-hydrogen-bondable methylthio group of MMTS is much smaller than that of the ethylsuccinimide group of NEM, modification of Cys(56) inhibited the interconversion between the m- and c-state conformation. The mechanism by which MMTS inhibited the transport activity is discussed in terms of stabilization of conformation of the loop M1.
Hashimoto
M
M
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-Tokushima 770-8505, Japan.
Majima
E
E
Hatanaka
T
T
Shinohara
Y
Y
Onishi
M
M
Goto
S
S
Terada
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000502:physiology*; D008956:Models, Chemical; D010618:Phenanthrolines / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013997:Time Factors
D003432:Cross-Linking Reagents; D010618:Phenanthrolines; D013439:Sulfhydryl Reagents; C014741:bis(1,10-phenanthroline)copper(2+) ion; C014674:methyl methanethiosulfonate; D000244:Adenosine Diphosphate; D000226:Mitochondrial ADP, ATP Translocases; D008741:Methyl Methanesulfonate; D003545:Cysteine