Photoaffinity labeling of mitochondrial adenosine triphosphatase by [.alpha.-32P]2-azido ADP

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Abstract
2-Azidoadenosine 5''-diphosphate (2-azido-ADP) labeled with 32P in the .alpha.-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., and Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [.alpha.-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding f [.alpha.-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 .mu.M in the presence of MgCl2 and 30 .mu.M in the presence of ethylenendiaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [.alpha.-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [.alpha.-32P]-2-azido-ADP/mol of F1. Under these conditions, only the .beta. subunit was photolabeled. Covalent binding of one photolabel per .beta. subunit was ascertained by electrophoretic separation of labeled and unlabeled .beta. subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled .beta. subunit. Upon incubation for several hours at room temperature in the dark with saturating concentrations of [.alpha.-32P]-2-azido-ADP, the amount of bound radioactivity increased steadily, and at 20 h, it amounted to 4.3 mol/mol of F1. This slow binding was accompanied by release of endogenous ADP or ATP, so that the total amount of bound nucleotides never exceeded 6 mol/mol of F1. The slowly reacting [.alpha.-32P]-2-azido-ADP was found to bind covalently, upon photoirradiation, to the .alpha. and .beta. subunits, with net predominance for the .beta. subunits possibly because of the orientation of the probe. Mapping studies indicated different patterns of photolabeling of the .beta. subunit after short and long periods of preincubation with [.alpha.-32P]-2-azido-ADP. These results are compatible with the presence of three loose nucleotide binding sites and three tight nucleotide binding sites per mitochondrial F1; the loose sites would be located on the .beta. subunits and the tight sites at the junction of the .alpha. and .beta. subunits.

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