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首页> 外文期刊>Biochemistry >Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: Identification of the transmembrane domain as a site for oligomerization
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Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: Identification of the transmembrane domain as a site for oligomerization

机译：野生型和跨膜结构域缺失的脂肪酸酰胺水解酶的比较表征：跨膜结构域作为寡聚化位点的鉴定

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Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (Delta TM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and Delta TM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and Delta TM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than Delta TM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for Delta TM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain-GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution. [References: 53]
机译：脂肪酸酰胺水解酶（FAAH）是一种完整的膜蛋白，负责水解许多伯和仲脂肪酸酰胺，包括神经调节化合物anandamide和oleamide。对FAAH一级序列的分析揭示了该酶的N末端存在单个预测的跨膜结构域。产生了缺少该N端跨膜结构域（Delta TM-FAAH）的大鼠FAAH蛋白的突变形式，并且发现它与野生型FAAH（WT-FAAH）一样，在COS-7细胞中表达时与膜紧密结合。 。从大肠杆菌表达和纯化的WT-和Delta TM-FAAH的重组形式表现出基本相同的酶学性质，也与大鼠肝脏的天然酶相似。通过化学交联，沉降速度分析超速离心和尺寸排阻色谱分析WT-和Delta TM-FAAH的低聚状态，表明两种酶在膜结合时和溶解后都是低聚的。然而，WT-FAAH始终表现出比Delta TM-FAAH更大的低聚物。此外，重组蛋白的SDS-PAGE分析确定了WT-FAAH而不是Delta TM-FAAH的SDS抗性寡聚物的存在。通过FAAH跨膜结构域-GST融合蛋白进一步证明了通过FAAH跨膜结构域的自缔合，该融合蛋白在溶液中形成了抗SDS的二聚体和大的寡聚体组装体。 [参考：53]

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