CARNITINE TRANSPORT IN NATURAL AND ARTIFICIAL MEMBRANES

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The long term objectives of this proposal are to elucidate the mechanism of acylcarnitine translocation across the mitochondrial inner membrane and to determine factors which interact with the translocase and control acylcarnitine cellular distribution. The overall specific aims are to determine structural and functional properties of the translocase isolated from beef heart submitochondrial particles. The translocase will be purified on a carnitine affinity column and activity reconstituted into phospholipid vesicles. Physical characterization of the isolated protein(s) will include: molecular weight, subunit composition, inhibitor-binding peptides, stoichiometry and affinity of substrate sites; as well as the transbilayer orientation in the presence or absence of substrate using the hydrophilic surface probe, 3-Azido-2,7-Naphthalene Disulfonate. Exchange and/or unidirectional transport activities will be determined. Reconstitution of the translocase will be otpimezed for phospholipid class and acyl chain saturation. The effect of the translocase on intervesicular transfer of acylcarnitine and interaction with fatty acid binding protein will be determined using the concentration sensitive probe, pyrenedodecanoyl-carnitine. Finally, the functional relationship of the translocase with purified carnitine palmitoyltransferase will be determined in phospholipid vesicles by fluorescent energy transfer techniques and by determination of [14C] carnitine release from translocated palmitoyl[14C]carnitine. Studies on the purified translocase are relevant to disease-associated impairment in either the translocase or translocase/transferase coupling, e.g., lipid storage myopathies, muscle dystrophy and certain cardiomyopathies.