THE RECONSTITUTED ISOLATED UNCOUPLING PROTEIN IS A MEMBRANE-POTENTIAL DRIVEN H+ TRANSLOCATOR
THE RECONSTITUTED ISOLATED UNCOUPLING PROTEIN IS A MEMBRANE-POTENTIAL DRIVEN H+ TRANSLOCATOR
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DOI：
10.1002/j.1460-2075.1985.tb04049.x
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发表时间：
1985-01-01
期刊：
影响因子：
11.4
通讯作者：
WINKLER, E
中科院分区：
文献类型：
--
作者：
KLINGENBERG, M;WINKLER, E
从棕色脂肪组织线粒体中分离的解偶联蛋白（UCP）被重组到人工磷脂囊泡中。由于H+运输的高度不稳定性，在重建中引入了几个新的步骤;洗涤剂辛基-POE，在溶解和纯化之前向线粒体中加入磷脂，通过用聚苯乙烯珠快速除去洗涤剂和通过混合离子交换除去外部盐来形成囊泡。在K+负载的蛋白脂质体中，H+内流可以通过添加缬氨霉素的扩散电位诱导。在UCP活性测定中，加入GTP可将H+内流抑制至90%以上。通过反转Δ。psi.然而，在外部K+、H+流出的情况下，以低四倍的速率测量。在囊泡装载有内部GTP，H+内流完全抑制，但可以激活Dowex-OH治疗，甚至更高的速度比发现在GTP-自由囊泡。与GTP的结合研究表明，大多数活性UCP与线粒体中的结合位点外部定向，并且在负载GTP的囊泡中，GTP也在外部结合。H+转运速率与膜电位呈线性关系。尽管有有序的取向，但没有“阀”机制，因为存在具有反向电势的H+流出。pH依赖性仅在pH 6.5和7.5之间很小，表明H+易位位点不同于高度pH依赖性的核苷酸结合位点。重组UCP的营业额与线粒体功能相当，并表明载体，而不是通道型H+运输。因此，UCP是迄今已知的最简单的H+转运蛋白。
The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing .DELTA..psi. with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no ''valve'' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport. The UCP is thus the simplest H+ transporter yet known.