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 it has been referred to as Ca2+-dependent NAT. On the other hand, members of the phospholipase A/acyltransferase (PLA/AT) 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 PLA/AT-2, and (3) NAT assay using radiolabeled substrate.