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首页> 外文期刊>The FEBS journal >Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane
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Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane

机译：纯化的RPE65与磷脂膜重新缔合后显示异构酶水解活性

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摘要

Generation of 11-cis-retinol from all-trans-retinyl ester in the retinal pigment epithelium is a critical step in the visual cycle and is essential for perception of light. Recent findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the reaction. However, previous attempts to detect the enzymatic activity of purified RPE65 were unsuccessful, and thus its enzymatic function remains controversial. Here, we developed a novel liposome-based assay for isomerohydrolase activity. The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing liposomes and demonstrated a robust isomerohydrolase activity. Furthermore, we found that all-trans-retinyl ester must be incorporated into the phospholipid membrane to serve as a substrate for isomerohydrolase. This assay system using purified RPE65 enabled us to measure kinetic parameters for the enzymatic reaction catalyzed by RPE65. These results provide conclusive evidence that RPE65 is the isomerohydrolase of the visual cycle.
机译：从视网膜色素上皮中的全反式视黄酯中生成11-顺式视黄醇是视觉周期中的关键步骤，对于感知光至关重要。细胞培养模型的最新发现表明，蛋白RPE65是催化该反应的类维生素A异构酶。然而，先前检测纯化的RPE65的酶促活性的尝试是失败的，因此其酶促功能仍存在争议。在这里，我们开发了一种新型的基于脂质体的异构水解酶活性测定方法。结果表明，纯化的重组鸡RPE65对含全反式棕榈酸棕榈酸酯的脂质体具有高亲和力，并显示出强大的异构水解酶活性。此外，我们发现必须将所有反式维甲酸酯掺入磷脂膜中，以用作异构水解酶的底物。使用纯化的RPE65的该测定系统使我们能够测量RPE65催化的酶促反应的动力学参数。这些结果提供了确凿的证据，证明RPE65是视觉周期的异构水解酶。

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