Reconstitution of recombinant uncoupling proteins -: UCP1,-2, and-3 have similar affinities for ATP and are unaffected by coenzyme Q10
Reconstitution of recombinant uncoupling proteins -: UCP1,-2, and-3 have similar affinities for ATP and are unaffected by coenzyme Q10
复制标题
DOI：
10.1074/jbc.m302126200
复制
发表时间：
2003-07-11
影响因子：
4.8
通讯作者：
Garlid, KD
中科院分区：
文献类型：
--
作者：
Jaburek, M;Garlid, KD
重组表达和重组方案的成功开发使解偶联蛋白（UCP）的转运特性和调节的详细研究成为可能。我们优化了细菌表达的解偶联蛋白的分离和重折叠条件，并重新检查了细菌表达的UCP 1，-2和-3在脂质体中重组的转运特性和调节。我们第一次发现ATP以相似的亲和力抑制UCP 1，-2和-3。在pH 7.2时，ATP抑制的Ki值为50 μ M（UCP 1）、70 μ M（UCP 2）和120 μ M（UCP 3）。这些对ATP的亲和力类似于用从棕色脂肪组织线粒体分离的天然UCP 1获得的亲和力（Ki = 65 μ M，pH 7.2）。质子传输的V-max值在UCPs中也相似，范围为8 - 20 μ mol.min（-1）.mg（-1），取决于实验条件。我们还研究了辅酶Q对含有重组UCP 1，- 2和- 3的脂质体中脂肪酸催化的质子通量的影响。我们发现辅酶Q对任何UCPs催化的脂肪酸依赖性质子转运没有影响，也不影响UCPs的核苷酸调节。我们的结论是辅酶Q是不是一个辅因子的UCP介导的质子运输。
The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, - 2, and - 3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, - 2, and - 3 with similar affinities. The K-i values for ATP inhibition were 50 muM (UCP1), 70 muM (UCP2), and 120 muM (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (K-i = 65 muM at pH 7.2). The V-max values for proton transport were also similar among the UCPs, ranging from 8 to 20 mumol.min(-1).mg(-1), depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, - 2, and - 3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport.