Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate

Abstract

2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.