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CARNITINE TRANSPORT IN NATURAL AND ARTIFICIAL MEMBRANE

天然和人造膜中肉碱的转运

基本信息

批准号：
3354744
负责人：
PAUL E WOLKOWICZ
金额：
$ 9.15万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-08-01 至 1990-07-31
项目状态：
已结题

项目摘要

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.

这项建议的长期目标是阐明酰肉碱在线粒体内膜上的易位确定与转位酶相互作用的因素和控制酰卡尼汀的细胞分布。总体的具体目标是确定分离的转位酶的结构和功能性质来自牛心的亚线粒体颗粒。转位酶将是在肉碱亲和层析柱上纯化，并将活性重组为磷脂小泡。孤立的物理特征蛋白质(S)将包括：分子量、亚基组成、抑制剂结合肽、化学计量比和底物位置的亲和力；以及在存在或不存在的情况下的跨双层取向使用亲水性表面探针3-叠氮基-2，7-萘的底物二磺酸盐。交换和/或单向运输活动将下定决心。转位酶的重建将被优化为磷脂类和酰链饱和度。美国政府的影响转位酶在酰肉碱囊间转移中的作用及相互作用用与脂肪酸结合的蛋白质的浓度来测定灵敏的探针，吡喃十二烷基肉碱。最后，功能转位酶与提纯肉碱的关系磷脂小泡中棕榈酰基转移酶的测定荧光能量转移技术和[14C]的测定从转位的棕榈酰[14C]肉碱释放肉碱。研究：关于纯化的转位酶与疾病相关的损害有关转位酶或转位酶/转移酶偶联，例如脂类储藏性肌病、肌肉营养不良和某些心肌病。