Isolation of the unliganded adenosine 5'-diphosphate,adenosine 5'-triphosphate carrier-linked binding protein and incorporation into the membranes of liposomes
The ADP,ATP carrier-linked binding protein of beef heart mitochondria was isolated free of ligands, using the detergent 3-lauramido-N,N-dimethylpropylamine oxide. Unlike the preparation of the carboxyatractylate (CAT)-protein complex described earlier, this protein enables direct binding studies to be made with the inhibitor ligands. The protein was characterized with respect to its polypeptide composition, stability against degradation, and immunological properties; the identity of the binding protein with the previously isolated CAT-protein complex was thereby shown. As a step toward reconstitution studies, the isolated binding protein was incorporated into liposomes by a simple rapid mixing process. The complete insertion into the vesicular membrane was demonstrated by chromatography on Sepharose 6B and by immunoprecipitation reactions.