Extraction and partial purification of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase from rat liver microsomes

Abstract

Acyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) was extracted from rat liver microsomes with an aqueous dispersion of 1-acyl-sn-glycero-3-phosphocholine, a substrate of the enzyme, and purified up to 30-fold. The procedure includes removal of unrelevant proteins and lipids by washings of microsomes with a buffer of high ionic strength and with buffers containing detergents, extraction of the enzyme with an aqueous dispersion of 1-acyl-sn-glycero-3-phosphocholine, and chromatography by gel filtration. The acyltransferase was eluted from a Ultrogel AcA 34 column at a position with a Kav of 0.122; an elution position of a protein with a molecular weight of 225 000. The partially purified enzyme was active over a wide range of pH with an optimum at around pH 8. Depending on the acyl donors, different rates of the reaction were obtained by the preparation. The order was: arachidonoyl-CoA greater than linoleoyl-CoA = oleoyl-CoA greater than palmitoyl-CoA. The enzyme preparation acylated 1-acyl-sn-glycero-3-phosphocholine, 1-acyl-sn-glycero-3-phospho-ethanolamine and 1-acyl-sn-glycero-3-phosphoinositol but not acylated 2-acyl-sn-glycero-3-phosphocholine, 1-acyl-sn-glycerol 3-phosphate or diacylglycerol. Some sulfhydryl-binding reagents inactivated the enzyme.