Photoaffinity labeling of the adenine nucleotide carrier in heart and yeast mitochondria by an arylazido ADP analog

Abstract

▪ 1. 1. Arylazido analogs of ADP and ATP ( N-4-azido-2-nitrophenyl-aminobutyryl-ADP and N-4-azido-2-nitrophenylaminobutyryl-ATP have been prepared in radioactive form and used in photolabeling experiments to identify the adenine nucleotide carrier in mitochondria and sonic submitochondrial particles. 2. 2. When added in the dark to beef heart mitochondria, azidonitrophenyl-aminobutyryl-ADP binds to the adenine-nucleotide carrier. It is not transported across the membrane to the matrix space, but it inhibits ADP transport in mito-chondria. The inhibition is of a mixed type with a K i value of about 10 μM. 3. 3. The nitrene derivative formed upon photoirradiation of tritiated azidonitrophenylaminobutyryl-ADP or -ATP binds to a polypeptide of apparent molecular weight 30 000 in beef heart mitochondria and 37 000 in Saccharomyces cerevisiae mitochondria. The photolabeling is prevented by preincubation of the mitochondria with atractyloside or carboxyatractyloside. 4. 4. Photoirradiation of sonic submitochondrial particles from beef heart (inside-out particles) with tritiated azidonitrophenylaminobutyryl-ADP or -ATP results in the labeling of the 30 000-dalton polypeptide and also in the labeling of higher molecular weight peptides (50 000–55 000) probably belonging to F 1-ATPase. Addition of bongkrekic acid specifically decreases the photolabeling of the 30 000-dalton polypeptide. 5. 5. An arylazido derivative of atractyloside ( N-4-azido-2-nitrophenylaminobutyryl atractyloside) binds upon photoirradiation to the 30 000-dalton polypeptide in beef heart mitochondria and to the 37 000-dalton polypeptide in S. cerevisiae mitochondria. 6. 6. Since the adenine nucleotide carrier is readily damaged by ultraviolet light, nitro-arylazido analogs of ADP and ATP or of atractyloside, which are photoactivated in visible light, were used in preference to other azido analogs, which require ultraviolet light for photoactivation. 7. 7. Data presented in this paper support the view that the same mitochondrial protein belonging to the adenine nucleotide transport system is able to bind ADP (or ATP) and atractyloside.