Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids

In this study, we investigated how the nature of the phospholipid head grou p and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin ch olesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebu tanoyl)-phosphatidylcholine, 1,2-bis-(1-pyrenebutanoyl)phosphatidylethanola mine and 1,2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monome rs or incorporated into small unilamellar vesicles consisting of dipalmitoy lphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled ph ospholipids was determined both by fluorescence and by high performance liq uid chromatography. V-max and K-m were calculated for the different substra tes. The data show that V-max is 10- to 30-fold higher for the hydrolysis o f monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine ( PE) and the phosphatidylalcohols, while K-m values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholi ne ether vesicles, we observed a 4- to 10-fold increase of V-max for PE and the phosphatidylalcohols, and no significant change for K-m. V-max for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity o n monomeric PC and PE. These data suggest that the activity of LCAT is dete rmined both by the molecular structure of the phospholipid and by its macro molecular properties. The LCAT activity on monomeric substrates decreases a s: phosphatidylcholine >> phosphatidylethanolamine congruent to phosphatidy lpropanol congruent to phosphatidylethanol congruent to phosphatidylethylen eglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospho lipid head group. (C) 2000 Elsevier Science B.V. All rights reserved.