Nonenzymatic proton handling by carbonic anhydrase II during H+-lactate cotransport via monocarboxylate transporter 1
Nonenzymatic proton handling by carbonic anhydrase II during H+-lactate cotransport via monocarboxylate transporter 1
复制标题
DOI：
10.1074/jbc.m802134200
复制
发表时间：
2008-08-01
影响因子：
4.8
通讯作者：
Deitmer, Joachim W.
中科院分区：
文献类型：
--
作者：
Becker, Holger M.;Deitmer, Joachim W.
碳酸酐酶（Carbonic anhydrase，CA）是一种普遍存在的催化二氧化碳、质子和碳酸氢盐平衡的酶。对于几种酸/碱偶联的膜载体，已经表明CA的催化活性支持转运活性，这种相互作用被称为“转运代谢子”。“我们已经报道了CA亚型II（CAII）增强了在非洲爪蟾卵母细胞中表达的单羧酸转运体亚型I（MCT 1）的乳酸转运活性，这不需要CAII催化活性（Becker，H. M.，Fecher-Trost，C.，Hirnet，D.，Sultemeyer，D.，和Deitmer，J.W.（2005）J.Biol.Chem.280，39882-39889）。MCT 1与野生型CAII或无催化活性的突变体CAII-V143 Y的共表达同样增强了MCT 1的活性，尽管注射CAI或CAII的N-末端突变体的共表达对MCT 1的转运活性没有影响，证明了CAII对通过MCT 1的乳酸转运的特异性非酶促作用。如果通过在高H+浓度下施加低浓度的乳酸盐，将H+梯度设置为主导乳酸盐转运速率，则CAII的效果最大。我们通过测量局部应用乳酸盐期间不同感兴趣区域中荧光染料的pH变化来测试CAII是否有助于H+沿着细胞膜内表面穿梭的假设。细胞内pH值的变化衰减从乳酸盐应用的焦点到更远的网站少得多时，CAII已被注射。我们提出了一个假设的模型，其中的有效移动的H+到散装胞质溶胶增加CAII，从而减缓耗散的H+梯度穿过细胞膜，这驱动MCT 1活性。
Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the equilibration of carbon dioxide, protons, and bicarbonate. For several acid/base-coupled membrane carriers it has been shown that the catalytic activity of CA supports transport activity, an interaction coined "transport metabolon." We have reported that CA isoform II (CAII) enhances lactate transport activity of the monocarboxylate transporter isoform I (MCT1) expressed in Xenopus oocytes, which does not require CAII catalytic activity (Becker, H. M., Fecher-Trost, C., Hirnet, D., Sultemeyer, D., and Deitmer, J. W. (2005) J. Biol. Chem. 280, 39882-39889). Coexpression of MCT1 with either wild type CAII or the catalytically inactive mutant CAII-V143Y similarly enhanced MCT1 activity, although injection of CAI or coexpression of an N-terminal mutant of CAII had no effect on MCT1 transport activity, demonstrating a specific, nonenzymatic action of CAII on lactate transport via MCT1. If the H+ gradient was set to dominate the rate of lactate transport by applying low concentrations of lactate at a high H+ concentration, the effect of CAII was largest. We tested the hypothesis of whether CAII helps to shuttle H+ along the inner face of the cell membrane by measuring the pH change with fluorescent dye in different areas of interest during focal lactate application. Intracellular pH shifts decayed from the focus of lactate application to more distant sites much less when CAII had been injected. We present a hypothetical model in which the effective movement of H+ into the bulk cytosol is increased by CAII, thus slowing the dissipation of the H+ gradient across the cell membrane, which drives MCT1 activity.