Stoicheiometry of dicyclohexylcarbodiimide-ATPase interaction in mitochondria.
1. The oligomeric dicyclohexylcarbodiimide (DCCD)-binding protein of mitochondrial ATPase was studied using (a) the relationship between (14C] DCCD binding and inhibition of ATPase activities and (b) the analysis of the kinetics of inhibition. 2. The [14C]DCCD binding to bovine heart mitochondria is linearly proportional to the inhibition of ATP hydrolysis up to a 50% decrease of the original activity resulting in 0.6 mol DCCD bound covalently to the specific inhibitory site (Houstĕk, J., Svoboda, P., Kopecký, J., Kuzela, S. and Drahota, Z. (1981) Biochim. Biophys. Acta 634, 331-339) per mol of the fully inhibited enzyme. 3. Kinetics of the inhibition of both the ATPase activity (heart and liver mitochondria) and ADP-stimulated respiration (liver) reveal that 1 mol DCCD per mol ATPase eliminates both the synthetic and the hydrolytic activities. It is inferred that the activity-binding correlation underestimates that number of DCCD-reactive sites. 4. The second-order rate constant of the DCCD-ATPase interaction (k) is inversely related to the concentration of membranes, indicating that DCCD reaches the inhibitory site by concentrating in the hydrophobic (phospholipid) environment. 5. At a given concentration of liver mitochondria, comparable k values are obtained both for the inhibition of ATP hydrolysis (k = 5.35.10(2)M-1.min-1) and ADP-stimulated respiration (k = 5.67.10(2)M-1.min-1). 6. It is concluded that both the synthetic and the hydrolytic functions if ATPase are inhibited via a common single DCCD-reactive site. This site is represented by one of the several polypeptide chains forming the oligomer of the DCCD-binding protein. The inhibitor-ATPase interaction does not exhibit cooperativity, indicating that the preferential reactivity towards DCCD is an inherent property of the inhibitory site.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002234:Carbodiimides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008929:Mitochondria, Heart / Q000378:metabolism*
D002234:Carbodiimides; D002352:Carrier Proteins; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases