Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel
Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel
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胞浆苹果酸脱氢酶赋予核酸传导通道选择性
DOI：
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发表时间：
2002
影响因子：
11.1
通讯作者：
P. Klotman
中科院分区：
文献类型：
--
作者：
B. Hanss;E. Leal;A. Teixeira;Robert E. Christian;Jeffery Shabanowitz;D. Hunt;P. Klotman
我们先前已经描述了对核酸具有高度选择性的细胞表面通道。核酸电导率为10 pS，该通道对寡脱氧核苷酸的选择性比任何测试的阴离子至少高10，000倍（1）。在此，我们提供的证据表明，核酸传导通道（NACh）是一个异源多聚体复合物的至少两种蛋白质;一个45 kDa的孔形成亚基（p45）和一个36 kDa的调节亚基（p36）。p45在平面脂质双层中的重构导致通道的形成，该通道在不存在核酸的情况下门控，并且对阴离子（包括寡核苷酸）比阳离子更具选择性。该通道表现出从一个电流水平到另一个电流水平（或到闭合状态）的转变;然而，转变的发生率很低。当p36单独重建时，未观察到通道活性。用p45重建p36恢复了对通道的核酸依赖性和选择性。蛋白质序列分析鉴定p36为胞质苹果酸脱氢酶（cMDH）。进行实验以证明cMDH是NACh的调节亚基。当用猪心cMDH而不是线粒体MDH重建p45时，观察到选择性活性。酶底物l-苹果酸和针对cMDH产生的抗血清均阻断NACh活性。这些数据表明，核酸传导通道是至少两种蛋白质p45和cMDH的复合物。此外，这些数据确定cMDH赋予通道的核酸选择性。
We have described previously a cell surface channel that is highly selective for nucleic acids. Nucleic acid conductance is 10 pS and the channel is at least 10,000-fold more selective for oligodeoxynucleotides than any anion tested (1). Herein we provide evidence that the nucleic acid-conducting channel (NACh) is a heteromultimeric complex of at least two proteins; a 45-kDa pore-forming subunit (p45) and a 36-kDa regulatory subunit (p36). Reconstitution of p45 in planar lipid bilayers resulted in formation of a channel which gated in the absence of nucleic acid and which was more selective for anions (including oligonucleotide) than cations. This channel exhibited transitions from one level of current to another (or to the closed state); however the incidence of transitions was rare. Channel activity was not observed when p36 was reconstituted alone. Reconstitution of p36 with p45 restored nucleic acid dependence and selectivity to the channel. Protein sequence analysis identified p36 as cytosolic malate dehydrogenase (cMDH). Experiments were performed to prove that cMDH is a regulatory subunit of NACh. Selective activity was observed when p45 was reconstituted with pig heart cMDH but not with mitochondrial MDH. Both the enzyme substrate l-malate and antiserum raised against cMDH block NACh activity. These data demonstrate that a nucleic acid conducting channel is a complex of at least two proteins, p45 and cMDH. Furthermore, these data establish that cMDH confers nucleic acid selectivity of the channel.
DOI：
10.1073/pnas.95.4.1921
发表时间：
1998-02-17
影响因子：
11.1
作者：
Hanss, B;Leal-Pinto, E;Klotman, PE
通讯作者：
Klotman, PE