Interaction between spin-labeled acyl-coenzyme A and the mitochondrial adenosine diphosphate carrier

Spin-labeled long-chain (m,n)acyl-CoA's (general formula: CH3(CH2)mCR(CH2)nCOSCoA, where R is an oxazolidine ring containing a nitroxide) inhibit anion transports through the inner mitochondrial membrane at low concentrations as ordinary long-chain acyl-CoA's do. The inhibition constant relative to the inhibition of the ADP transport in heart mitochondria by spin-labeled palmityl-CoA and stearyl-CoA is of the order of 10-7 M, a value which is similar to that found for natural long-chain acyl-CoA's. A short-chain spin-labeled acyl-CoA (C5) showed no inhibitory effect in the range of concentrations tested (up to 30 muM). (2) (10,3)Acyl-CoA added to heart mitochondria at low concentrations exhibits spectra corresponding to an immobilized probe. The corresponding free fatty acid shows a higher freedom of motion between 0 and 30 degrees. The same differences in spectra of spin-labeled acyl-CoA and spin-labeled free fatty acid were found in inner membrane vesicles from rat liver mitochondria, but not in outer membrane preparations. (3) The selective interaction of spin-labeled acyl-CoA with the ADP carrier is indicated by the release of this interaction by specific ligands of the ADP carrier, such as ADP or ATP, carboxyatractyloside, adn bongkrekic acid. ADP (or ATP) and carboxyatractyloside rendered the spin-labeled (10,3)acyl-CoA nearly as mobile as the (10,3) free fatty acid. No effect was obtained with AMP, GDP, or UDP which are not transported by the ADP carrier. Bongkrekic acid, another specific inhibitor of the ADP carrier, was inactive when added alone; however, it was effective when added together with amounts of ADP which are ineffective per se. (4) The electron spin resonance (esr) spectrum observed at low concentrations of (10,3)acyl-CoA arises from (10,3)acyl-CoA bound to the ADP carrier. At higher concentrations the (10,3)-acy-CoA is more suggesting that the bulk of the label is also present in the lipid phase of the membrane. Spin-labeled acylCoA's incorporated into a sonicated dispersion of lipids extracted from heart mitochondria exhibited similar mobile spectra. (5) When the oxazolidine ring is moved down the hydrocarbon chain of the acyl-CoA, the binding features tended to disappear. Whereas nitroxide-protein interactions could be easily measured with the (10,3)acyl-CoA and the (7,6)acyl-CoA, much less or even no significant interactions could be detected with the (5,10)acyl-CoA or the (1,14)acyl-CoA. (6) The above results suggest that spin-labeled long-chain acylCoA added to mitochondria binds by its polar moiety to the ADP carrier. The acyl chain interacts with the ADP carrier protein over a length of 10-15 A. The remaining portion of the acyl chain experiences a fluid lipid environment.