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Exploration of nucleotide binding sites in the mitochondrial membrane by 2‐azido‐[α‐ 32 P]ADP
Author(s) -
Dalbon Pascal,
Boulay François,
Vignais Pierre V.
Publication year - 1985
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(85)81073-5
Subject(s) - submitochondrial particle , atp–adp translocase , atpase , mitochondrion , adenosine diphosphate , biochemistry , chemistry , nucleotide , adenine nucleotide , protein subunit , binding site , inner mitochondrial membrane , affinity label , adenosine triphosphate , membrane transport , biophysics , membrane , biology , enzyme , platelet aggregation , platelet , gene , immunology
The ADP/ATP carrier of beef heart mitochondria is able to bind 2‐azido‐[α‐ 32 P]ADP in the dark with a K d value of ∼ 8 μM. 2‐Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2‐azido‐[α‐ 32 P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside‐out submitochondrial particles with 2‐azido‐[α‐ 32 P]ADP, both the ADP/ATP carrier and the β subunit of the membrane‐bound F 1 ‐ATPase are covalently labeled. The binding specificity of 2‐azido‐[α‐ 32 P]ADP for the β subunit of F 1 ‐ATPase is ascertained by prevention of photolabeling of isolated F 1 by preincubation with an excess of ADP.