Purification and characterization of a transmembrane domain-deleted form of lecithin retinol acyltransferase
Purification and characterization of a transmembrane domain-deleted form of lecithin retinol acyltransferase
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DOI：
10.1021/bi0342416
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发表时间：
2003-05-27
期刊：
影响因子：
2.9
通讯作者：
Rando, RR
中科院分区：
文献类型：
--
作者：
Bok, D;Ruiz, A;Rando, RR
卵磷脂视黄醇酰基转移酶（LRAT）催化全反式视黄醇酯化成全反式视黄醇酯，是脊椎动物视觉循环的重要反应。由于全反式视黄醇酯是产生11-顺式类视黄醇异构化反应的底物，因此这种酯化反应在视觉循环的操作中是必不可少的。此外，LRAT是一系列序列新颖、功能未知的蛋白的创始成员。原生LRAT是一种完整的膜蛋白，从未被纯化过。为了获得纯LRAT，将N-和c -跨膜末端删除并用poly His标记代替以进行纯化。这种截断形式的LRAT，称为tLRAT，已在细菌中表达并完全纯化。tLRAT具有催化活性，处理全反式视黄醇的效率至少是11-顺式视黄醇（视觉发色团的前体）的10倍。虽然tLRAT可以在细菌中稳定表达，但由于该酶仍然含有可能相互作用的疏水结构域，因此需要洗涤剂进行提取。事实上，tLRAT可以寡聚并形成二聚体。天然LRAT也形成功能性同二聚体。这些研究为大规模制备纯tLRAT铺平了道路，为进一步的机制和结构研究奠定了基础。
Lecithin retinol acyltransferase (LRAT) catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester, an essential reaction in the vertebrate visual cycle. Since all-trans-retinyl esters are the substrates for the isomerization reaction that generates 11-cis-retinoids, this esterification reaction is essential in the operation of the visual cycle. In addition, LRAT is the founder member of a series of proteins, which are of novel sequence and have unknown functions. Native LRAT is an integral membrane protein and has never been purified. To obtain a pure LRAT, the N- and C-transmembrane termini were deleted and replaced with a poly His tag for the purpose of purification. This truncated form of LRAT, referred to as tLRAT, has been expressed in bacteria and fully purified. tLRAT is catalytically active and processes all-trans-retinol at least 10-fold more efficiently than 11-cis-retinol, the precursor to the visual chromophore. While tLRAT can be robustly expressed in bacteria, it requires detergent for extraction, as the enzyme still contains hydrophobic domains, which may interact. Indeed, tLRAT can oligomerize and forms dimers. Native LRAT also forms functional homodimers. These studies pave the way for the preparation of large-scale amounts of pure tLRAT for further mechanistic and structural studies.