人辅酶II依赖性视黄醇脱氢_还原酶选择性翻译起始位点分析.docVIP

 人辅酶 II 依赖性视黄醇脱氢/还原酶选择性 翻译起始位点分析# 梁斌，宋旭红，黄东阳** 5 10 15 20 （汕头大学医学院细胞生物学教研室，广东 汕头 515041） 摘要：目的：探讨分析 NADP(H)依赖性视黄醇脱氢/还原酶（NRDR）mRNA 中两个可选择 的翻译起始位点的作用。方法：构建 NRDR、NRDR278 原核表达载体和 GFP-tagged 真核表 达载体，通过酶活力测定、免疫荧光定位检测，分析 NRDR 可能的翻译起始位点。结果： NRDR 与 NRDR278 对所检测的底物的还原活性无显著差异。NRDR278 N 末端的线粒体定 位信号序列可以引导 NRDR278 进入线粒体，而 C 端暴露的过氧化物酶体定位信号亦可引导 融合蛋白进入过氧化物酶体。而 GFP-tagged NRDR，因缺失 N 端的线粒体定位信号，而无 法定位到线粒体。结论：NRDR 的翻译可能起始于第一个翻译起始位点。 关键词：NADP(H)依赖性视黄醇脱氢/还原酶；选择性翻译起始位点；酶活力；蛋白质亚细 胞定位 中图分类号：Q71，Q291 Alternative translation initiation site of human NADP(H)-dependent retinol dehydrogenase/reductase LIANG Bin, SONG Xuhong, HUANG Dongyang (Department of Cell Biology, Shantou University Medical College, GuangDong ShanTou 515041) Abstract: Objective: To determine the alternatively translational initiation site of NADP(H)-dependent retinol dehydrogenase/reductase (NRDR). Methods: The prokaryotic and eukaryotic expression vectors of NRDR and NRDR278 were constructed. The catalytic activities of the NRDR and NRDR278 towards α-dicarbonyl compounds were detected. 25 30 35 40 Immunofluorescence was used to analyze the localization of NRDR from the two alternatively translational initiation site respectively. Results: There is no significant difference between the catalytic activity of the NRDR and NRDR278 towards α-dicarbonyl compounds. NRDR278 with GFP fused to its N terminal is localized in peroxisome, and NRDR278 with GFP fused to its C terminal is localized in mitochondria. Conclusion: The amino acids between the two TIS are worked as signal sequence but not involved in the formation of the function structure. NRDR278 could be localized to mitochondria, suggesting the translation might be initiated at the first AUG. Keywords: NRDR; alternatively translational initiation site; catalytic activity; subcellular localization 0 引言 NADP(H) 依 赖 性 视 黄 醇 脱 氢 / 还 原 酶 （ NADP(H)-dependent retinol dehydrogenase/ reductase, NRDR）是参与体内维甲酸合成代谢的催化酶[1-3]，具有很强的视黄醇氧化和视黄 醛还原活性。而人的 NRDR 主要催化一些外源性的化合物，特别是带有苯环的