Assay of NAT Activity
Assay of NAT Activity
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NAT 活性测定
DOI：
10.1007/978-1-0716-2728-0_17
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发表时间：
2023
影响因子：
--
通讯作者：
Ueda Natsuo
中科院分区：
文献类型：
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作者：
Uyama Toru;Ueda Natsuo
在动物组织中，N-酰基转移酶（NAT）催化生物活性N-酰基乙醇胺生物合成途径中的第一步反应，将供体磷脂（如磷脂酰胆碱）的n-1位酰基链转移到磷脂酰乙醇胺的氨基上，形成N-酰基磷脂酰乙醇胺。NAT长期以来被认为是由Ca 2+刺激的，因此被称为Ca 2+依赖性NAT。后来，这种酶被鉴定为cPLA 2 ε（也称为PLA 2G 4 E）。另一方面，磷脂酶A/酰基转移酶（PLAAT）家族（也称为HRAS样抑制因子家族）的成员显示出不依赖于Ca 2+的NAT活性。在本章中，我们描述了（1）从大鼠脑中部分纯化Ca 2+依赖的NAT，（2）纯化重组cPLA 2 ε和PLAAT-2，和（3）使用放射性标记底物的NAT测定。
In animal tissues,N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactiveN-acylethanolamines, in which an acyl chain is transferred from thesn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation ofN-acylphosphatidylethanolamine. NAT has long been known to be stimulated by Ca2+and hence referred to as Ca2+-dependent NAT. Later, this enzyme was identified as cPLA2ε (also referred to as PLA2G4E). On the other hand, members of the phospholipase A/acyltransferase (PLAAT) family (also known as HRAS-like suppressor family) show Ca2+-independent NAT activity. In this chapter, we describe (1) partial purification of Ca2+-dependent NAT from rat brain, (2) purification of recombinant cPLA2ε and PLAAT-2, and (3) NAT assay using radiolabeled substrate.