title: Exploration of the nucleotide binding sites of the isolated ADP/ATP carrier protein from beef heart mitochondria. 1. Probing of the nucleotide sites by Naphthoyl-ATP, a fluorescent nontransportable analogue of ATP.
abstract: The ADP/ATP carrier protein was extracted and purified from beef heart mitochondria, and its binding parameters with respect to 3'-O-naphthoyladenosine 5'-triphosphate (N-ATP), a fluorescent nontransportable analogue of ATP, were studied. The binding of N-ATP to the isolated carrier protein was accompanied by a decrease in fluorescence. Conversely, the release of bound N-ATP upon addition of carboxyatractyloside (CATR) or ATP resulted in a fluorescence increase. The bound N-ATP that was released upon addition of an excess of CATR or ATP was referred to as specifically bound N-ATP, i.e., N-ATP bound to the nucleotide sites of the carrier protein. Two classes of binding sites for N-ATP could be identified; the number of high-affinity sites (Kd less than 10 nM) was equal to the number of low-affinity sites (Kd = 0.45 microM). CATR behaved apparently as a noncompetitive inhibitor of the binding of N-ATP. The amount of N-ATP released increased linearly with the amount of CATR added, indicating an extremely high affinity of the carrier protein for CATR. The number of CATR binding sites was equal to half the total number of N-ATP binding sites (high- and low-affinity sites); at saturating concentrations of N-ATP, the binding of 1 mol of CATR resulted in the release of 2 mol of bound N-ATP, one from the high-affinity site and the other from the low-affinity site, showing unambiguously that each CATR site is interacting with a pair of probably interdependent N-ATP sites. A clear competition between N-ATP and ATP for binding to the carrier protein was demonstrated. The Kd values of the high- and low-affinity sites for ATP were less than 50 nM and 5 microM, respectively. In the presence of high concentrations of ATP, the two classes of N-ATP binding sites became indistinguishable, suggesting interconversion. It is proposed that the asymmetry in affinity for N-ATP binding is induced by the binding step itself, the carrier protein exhibiting a negative cooperativity for N-ATP binding.
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D005456:Fluorescent Dyes / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000032:analysis*; D000226:Mitochondrial ADP, ATP Translocases / Q000032:analysis* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D001278:Atractyloside; C037102:naphthoyl-ATP; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; C003853:carboxyatractyloside