Binding of the fluorescein derivative eosin Y to the mitochondrial ADP/ATP carrier: characterization of the adenine nucleotide binding site.
As the SH-reactive fluorescein derivative eosin-5-maleimide (EMA) specifically labels Cys159 in the second loop facing the matrix space (loop M2) of the ADP/ATP carrier in bovine heart submitochondrial particles [Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187], we studied the interaction of non-SH-reactive eosin Y, an analog of EMA, with the carrier under various conditions to characterize its binding. Eosin Y was found to inhibit ADP transport by binding to loop M2 in submitochondrial particles, but not in mitochondria. Its Ki for transport (0.33 microM) was found to be very similar to its Kd (0.53 microM) for specific binding to the carrier. Bound eosin Y was displaced by the transport substrates ADP and ATP, but not by untransportable GTP, suggesting that eosin Y bound to the specific binding site of ADP and ATP. The three-dimensional structure and electrostatic features of eosin Y were very similar to those of ADP, and the hydrophobic property and divalent charge of eosin Y were very important for its binding to the carrier. Based on these results, the features of the binding site of the transport substrates are considered.
Majima
E
E
Faculty of Pharmaceutical Sciences, University of Tokushima, Japan.
Yamaguchi
N
N
Chuman
H
H
Shinohara
Y
Y
Ishida
M
M
Goto
S
S
Terada
H
H
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000737:chemistry; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D008958:Models, Molecular; D009206:Myocardium / Q000378:metabolism; D055672:Static Electricity
D000227:Adenine Nucleotides; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; C033531:eosin maleimide; D000226:Mitochondrial ADP, ATP Translocases; D004801:Eosine Yellowish-(YS)