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Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP
Abstract:
The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]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-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.
Insights
Beef heart mitochondria
Area of Science:
- Biochemistry
- Mitochondrial Physiology
- Molecular Biology
Background:
- The ADP/ATP carrier is crucial for mitochondrial energy transport.
- Understanding its interaction with ligands is key to energy metabolism research.
Purpose of the Study:
- To investigate the binding and photolabeling characteristics of 2-azido-[alpha-32P]ADP with mitochondrial proteins.
- To identify proteins interacting with ADP analogs in beef heart mitochondria.
Main Methods:
- Binding assays with 2-azido-[alpha-32P]ADP.
- Photoaffinity labeling of beef heart mitochondria and submitochondrial particles.
- Inhibition studies using carboxyatractyloside and ADP.
Main Results:
- 2-azido-[alpha-32P]ADP binds to the ADP/ATP carrier with a Kd of ~8 microM and inhibits ADP transport.
- Photoirradiation predominantly labels the ADP/ATP carrier, an effect blocked by carboxyatractyloside.
- In inside-out submitochondrial particles, both the ADP/ATP carrier and the F1-ATPase beta subunit are labeled.
- Binding to F1-ATPase beta subunit is specific for ADP, confirmed by competition assays.
Conclusions:
- 2-azido-[alpha-32P]ADP serves as a photoaffinity probe for the ADP/ATP carrier.
- This probe also identifies interactions with the F1-ATPase beta subunit in submitochondrial particles.
- The findings elucidate protein interactions within the mitochondrial energy transduction system.