ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by phosphorus-31 nuclear magnetic resonance

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Abstract
An unusual binding of cardiolipin to the ADP/ATP carrier has been found, which is distinguished by the relatively large amount and by the tightness of binding. High-resolution 31P NMR studies on the detergent-solubilized ADP/ATP carrier from beef heart mitochondria revealed narrow signals from phosphatidylcholine and phosphatidylethanolamine and a broadened signal of 3-40-Hz line width, suggestive of cardiolipin. Line broadening of this magnitude is to be expected when tumbling of the whole protein-detergent micelle is the only source of P-spin-spin relaxation. Thus, a strong immobilization of the protein-bound cardiolipin is inferred. By sucrose density gradient centrifugation phosphatidylcholine and phosphatidylethanolamine were removed, while approximately 6 .+-. 1 molecules of cardiolipin remained tightly bound in the dimeric protein molecule. The cardiolipin binding was stable against treatment with sodium dodecyl sulfate (SDS) although release of the inhibitor carboxyatractyloside revealed at least partial protein denaturation. Ca2+ did not readily interact with the bound cardiolipin. Complete detachment of the bound phospholipid was achieved by a short heat pulse in the presence of SDS. Denaturation of the carrier protein by guanidinium chloride or NaClO4 also led to release of the bound phospholipid. Thus, different stages of protein denaturation must be envisaged.