CARNITINE TRANSPORT IN NATURAL AND ARTIFICIAL MEMBRANE

本建议的长期目标是阐明 酰基肉毒碱转运穿过线粒体内膜， 确定与转位酶相互作用的因素， 酰基肉毒碱细胞分布。 总体具体目标是 确定分离的转位酶的结构和功能特性 来自牛心亚线粒体颗粒 移位酶将是 在肉毒碱亲和柱上纯化，并将活性重构为 磷脂囊泡。 分离物的物理表征 蛋白质将包括：分子量，亚基组成， 底物结合肽、化学计量和底物位点的亲和力; 以及在存在或不存在 使用亲水性表面探针的底物，3-叠氮基-2，7-萘 二磺酸盐。 交换和/或单向运输活动将 测定 易位酶的重建将被优化， 磷脂类和酰基链饱和度。 的影响 转位酶对酰基肉毒碱囊泡间转运及相互作用 与脂肪酸结合蛋白的浓度将使用 灵敏探针pyrenedodecanoyl-carnitine。 最后，功能 转位酶与纯化肉毒碱的关系 棕榈酰转移酶将在磷脂囊泡中测定， 荧光能量转移技术和[14 C]测定 从转位的棕榈酰[14 C]肉碱释放肉碱。 研究 纯化易位酶与疾病相关的损伤有关， 易位酶或易位酶/转移酶偶联，例如，脂质 存储性肌病、肌肉营养不良和某些心肌病。

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.