Interaction of 3'-O-(1-naphthoyl)adenosine 5'-diphosphate, a fluorescent adenosine 5'-diphosphate analogue, with the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier protein in the mitochondrial membrane.

Abstract

3'-O-(1-Naphthoyl)adenosine 5'-diphosphate (N-ADP), a fluorescent analogue of ADP, was established as a potent inhibitor of adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) transport in mitochondria and inside-out sonic particles; the Ki value was about 5 microM. The inhibition was of a mixed type. On the other hand, N-ADP was not transported in a measurable way in either type of particles. Upon binding to the particles, the fluorescent intensity of N-ADP was decreased; the release of the bound N-ADP upon addition of carboxyatractyloside (CATR) to mitochondria and bongkrekic acid (BA) to sonic particles was reflected by increases of fluorescence. In parallel assays dealing with 14C-labeled N-ADP, specifically bound [14C]N-ADP was equated to [14C]N-ADP released upon addition of either CATR (mitochondria) or BA (sonic particles). The specific binding of N-ADP corresponded to 1.4-1.6 nmol/mg of protein in mitochondria, with a Kd value of 3 microM, and to 1.5-1.6 nmol/mg of protein in sonic particles, with a Kd value of 6 microM. Essentially similar values were obtained for N-ATP binding. These values are at least twice as high as those found for specific ADP or ATP binding, suggesting that N-ADP or N-ATP binds to potential nucleotide binding sites that were not totally occupied by ADP or ATP. Whereas nearly all the specifically bound N-ADP in mitochondria was displaced by an excess of ADP (400 microM) at pH 7.4, only 30% could be removed from sonic particles under the same conditions. Furthermore at pH 6.5, no more than half of the specifically bound N-ADP could be removed by excess ADP in mitochondria and only 10-20% in sonic particles. These results indicate that each ADP/ATP carrier unit contains at least two types of nucleotide sites capable of interacting with N-ADP. Because of the hydrophobic nature of the naphthoyl moiety of N-ADP, the data suggest that difference in N-ADP binding in mitochondria and sonic particles are related to differences in the hydrophobic nature of their sites. Due to the special features of N-ADP (strong specific binding to the ADP/ATP carrier and no competence for transport), this DP analogue was particularly suitable for investigating the sensitivity of the nucleotide binding sites of the carrier to chemical modifiers. Inactivation studies were therefore carried out with mitochondria and sonic particles to compare the sensitivity to UV light and butanedione of the binding of N-ADP, [3H]BA, and [14C]Ac-CATR, a radiolabeled substitute for CATR. Both in mitochondria and in sonic particles, UV light and butanedione inactivated more rapidly the binding of N-ADP than that of [3H]BA. On the other hand, in mitochondria, UV light inactivated more rapidly the binding of [14C]Ac-CATR than that of N-ADP; the reverse was true for the inactivation by butanedione. The inactivation data conclusively indicate that BA, CATR, and adenine nucleotides are recognized by different specific sets of amino acids.