RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle
RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle
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DOI：
10.1074/jbc.m508903200
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发表时间：
2006-02-03
影响因子：
4.8
通讯作者：
Ma, JX
中科院分区：
文献类型：
--
作者：
Moiseyev, G;Takahashi, Y;Ma, JX
在视网膜色素上皮(RPE)中，全反式视黄酸酯异构化为11-顺式视黄醇是视觉周期中的关键步骤，对正常视觉是必不可少的。最近，我们确定RPE65蛋白是催化这一反应的异构体水解酶。本研究调查了RPE65的异位氧化水解酶活性是否需要金属离子。以全反式[H-3]视黄醇转化为11-顺式-[H-3]视黄醇作为异麦芽水解酶活性的指标。金属螯合剂2，2‘-联吡啶和1，10-菲咯啉均呈剂量依赖性抑制牛RPE微粒体中异麦芽水解酶活性，IC50值分别为0.5和0.2 mM。在相同的反应体系中，卵磷脂-视黄醇酰基转移酶(LRAT)活性不受这些金属螯合剂的影响。被金属螯合剂抑制的异位水解酶活性可被FeSO4恢复，但不能被CuSO4、ZnCl2或MgCl2恢复。此外，添加Fe(III)柠檬酸盐或FeCl3均不能使酶活恢复，说明Fe2+是异麦芽水解酶活性必需的金属离子。为了证实这一结果，我们在稳定表达LRAT的293A细胞系中表达了RPE65。体外活性测定表明，两种金属螯合剂均能抑制重组RPE65的异构体水解酶活性。FeSO4的加入恢复了重组RPE65的酶活性。此外，两种特异的铁染色方法表明，纯化的RPE65含有内源性铁。电感耦合等离子体质谱测量表明，牛RPE65与化学计量比为0.8+/-0.1的铁离子结合。这些结果表明，RPE65在视觉周期中是一种铁依赖的异位水解酶。
The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[H-3] retinol to 11-cis-[H-3]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2'-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mM, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 +/- 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle.