KINETIC, BINDING AND ULTRASTRUCTURAL PROPERTIES OF THE BEEF-HEART ADENINE-NUCLEOTIDE CARRIER PROTEIN AFTER INCORPORATION INTO PHOSPHOLIPID-VESICLES
KINETIC, BINDING AND ULTRASTRUCTURAL PROPERTIES OF THE BEEF-HEART ADENINE-NUCLEOTIDE CARRIER PROTEIN AFTER INCORPORATION INTO PHOSPHOLIPID-VESICLES
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DOI：
10.1016/0005-2728(80)90103-6
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
VONVIGNAIS, P
中科院分区：
文献类型：
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作者：
BRANDOLIN, G;DOUSSIERE, J;VONVIGNAIS, P
通过将纯化的载体蛋白掺入充满 ATP 的脂质体中来重建 ADP/ATP 转运。通过将[14C]ADP摄取到脂质体中并通过发光技术测定的ATP释放来测定转运。 [14C]ADP 摄取严格依赖于内部 ATP。 ADP/ATP 转运通过 1/1 化学计量的交换扩散进行。 ADP 和 ATP 的特异性是绝对的。交换的容量和速率取决于脂质体中存在的 ATP 浓度。 20℃时的传输速率C，在 20 mM 内部 ATP 下，每分钟每毫克添加的载体蛋白交换的核苷酸量通常在 300-1000 nmol 之间。外部 ADP 的表观 Km 值约为 10 μM。 ADP/ATP 交换对于老化相当稳定。 20℃老化1天后仅下降20%。 C. 浓度 > 1-2 mM 的 Mg2+、Mn2+ 和 Ca2+ 对 ADP/ATP 转运具有有害影响，同时伴有内部 ATP 的释放和多层囊泡的积累。苍术苷表现为竞争性抑制剂，羧基苍术苷表现为非竞争性抑制剂。 Bongkrekic 酸需要弱酸性 pH 值才能发挥抑制作用。有关苍术苷、羧基苍术苷和白术酸的数据与用整个线粒体获得的数据相似，表明脂质体中的载体蛋白具有与线粒体中相同的不对称排列。根据有关苍术苷或羧基苍术苷对ADP/ATP转运的抑制的剂量反应数据以及由ADP去除的结合的[3H]-苍术苷的量计算脂质体中感受态载体蛋白的百分比。通过这两种方法，基于 1 个苍术苷或羧基苍术苷分子/30,000 MW 载体单位的结合导致运输完全抑制的假设，3-6% 的添加载体蛋白能够进行 ADP/ATP 运输。冷冻断裂电镜显示，ADP/ATP 载体蛋白-脂质制剂由小囊泡形成，其中大部分产生光滑的断裂面（可能是纯脂质囊泡）。只有一小部分囊泡（2-4%，取决于添加的载体蛋白的量）是明显颗粒化的。约 90% 的颗粒囊泡显示不超过 2 个颗粒/囊泡，仅 5% 的颗粒囊泡超过 5 个颗粒/囊泡。凸、凹断裂面间颗粒分布不对称；大约 2/3 的蛋白质分子锚定在囊泡的外表面，只有 1/3 锚定在内部。这种不对称分布在添加羧基苍术苷后没有显着改变，但在添加bongkrekic酸后发生了巨大变化，导致锚定在囊泡内表面的蛋白质分子百分比增加。 ADP/ATP 载体蛋白可能能够通过翻译穿过磷脂膜。数据的解释是假设载体在邦克酸结合时稳定在更暴露于内部的构象中，而在(羧基)白术苷结合时则稳定在更暴露于外部的构象中。 ADP/ATP 运输可能涉及类似的平移运动。
ADP/ATP transport was reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. ADP/ATP transport proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for ADP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20.degree. C, at 20 mM internal ATP, routinely ranged between 300-1000 nmol of nucleotide exchanged/min per mg of added carrier protein. The apparent Km value for external ADP was around 10 .mu.M. ADP/ATP exchange was rather stable to aging. It dropped by only 20% after 1 day of aging at 20.degree. C. Mg2+, Mn2+ and Ca2+ at concentrations > 1-2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in mitochondria. The percentage of competent carrier protein in liposomes was calculated from dose-reponse data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3-6% of the added carrier protein was competent in ADP/ATP transport, based on the assumption that the binding of 1 atractyloside or carboxyatractyloside molecule/30,000 MW carrier unit results in complete inhibition of transport. Freeze-fracture EM showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2-4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles/vesicle and only 5% more than 5 particles/vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2/3 of the protein molecules were anchored at the external surface of the vesicles and only 1/3 at the internal one. This asymmetric distribution was not significantly modified after the addition of carboxyatractyloside but changed drastically upon addition of bongkrekic acid, leading to an increased percentage of protein molecules anchored at the internal surface of the vesicles. The ADP/ATP carrier protein is probably able to move by translation across the phospholipid membrane. The data are interpreted by assuming that the carrier is stabilized in a conformation more exposed to the inside upon bongkrekic acid binding, and to the outside upon (carboxy)atractyloside binding. A similar translational motion could be involved in ADP/ATP transport.