Characterization by photoaffinity labeling of the adenine nucleotide carrier in plant mitochondria

1.

1. The covalent photolabeling of the adenine nucleotide carrier in potato mitochondria and bean mitochondria by photoactivable derivatives of atractyloside and ADP is described.

2.

2. In the absence of photoirradiation, N-4-azido-2-nitrophenylaminobutyryl atractyloside (arylazido atractyloside) was found to compete with atractyloside and ADP for binding to potato and bean mitochondria. Bound arylazido atractyloside was displaced by atractyloside and ADP.

3.

3. Upon incubation with potato or bean mitochondria and photoirradiation, [³H]arylazido atractyloside was found to bind covalently to a mitochondrial polypeptide of apparent molecular weight 35 000. Photolabeling by [³H]-arylazido atractyloside was prevented by preincubation of mitochondria with carboxyatractyloside or ADP.

4.

4. Photoirradiation with ³H-labeled N-4-azido-2-nitrophenylaminobutyryl ADP ([³H]arylazide ADP) also resulted in the covalent photolabeling of the 35 000 dalton polypeptide. Photolabeling by [³H]arylazido ADP was prevented by preincubation with carboxyatractyloside.

5.

5. The 35 000 dalton polypeptide covalently labeled by [³H]arylazido-atractyloside was isolated by hydroxyapatite gel chromatography and its homogeneity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

6.

6. The molecular weight of the adenine nucleotide carrier protein of potato and bean mitochondria (35 000) is close to that of Saccharomyces cerevisiae mitochondria (molecular weight 37 000) and significantly higher than that of mammalian mitochondria (molecular weight 30 000).

7.

7. The photolabeling data presented in this paper indicate that in spite of the fact that potato mitochondria bind atractyloside rather loosely, they possess a specific recognition site like bean mitochondria which bind atractyloside much more firmly. The same polypeptide, namely the adenine nucleotide carrier protein, is able to bind both ADP and atractyloside.