Assay of NAT Activity

Abstract

In animal tissues, N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactive N-acylethanolamines, in which an acyl chain is transferred from the sn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation of N-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.

Keywords: COS-7 cell; Ca-NAT; N-Acylphosphatidylethanolamine; N-Acyltransferase; NAPE; PLAAT family; Radioisotope; Rat brain; Thin-layer chromatography; cPLA2ε.