FORMATION OF VESICLES BY THE ACTION OF ACYL-COA-1-ACYLLYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE FROM RAT-LIVER MICROSOMES - OPTIMAL SOLUBILIZATION CONDITIONS AND ANALYSIS OF LIPID-COMPOSITION AND ENZYME-ACTIVITY

The enzyme acyl coenzyme A:1-acyllysophosphatidylcholine acyltransfera se (acyl-CoA:lysoPC acyltransferase) can be isolated in newly formed p hosphatidylcholine (PC) vesicles by solubilization of rat liver micros omes with the two substrates lysoPC and acyl-CoA. In this study, we so ught to optimize the conditions for the formation of PC vesicles and a nalyzed the lipid composition and enzyme activity of the newly formed vesicles. Analysis of PC vesicles formed by incubation of the microsom al preparation with 1-(C-16:0)lysoPC and C(18:1)CoA, C(18:2)CoA, or C( 20.4)CoA showed that the optimal protein:lysoPC ratio was 1:5 (by weig ht) and the optimal lysoPC:acyl-CoA ratio was 1:1 (molar amounts). PC formation increased with incubation time; after 20 h of incubation at 37 degrees C, approximately 75% of the lysoPC was converted to PC in t he incubation mixture. The phospholipid molecular species composition of the vesicles reflected almost exclusively the substrates used; the vesicles contained approximately 33% of the total acyl-CoA:lysoPC acyl transferase activity from the microsomes and demonstrated a single pro tein band with a molecular mass of 21 kDa by gel electrophoresis. The procedure selected for the enzyme specific for lysoPC acylation, as en zyme activity toward lysophosphatidylethanolamine (lysoPE), lysophosph atidylserine (lysoPS), and lysophosphatidylinositol (lysoPI), was very low. In addition, the utilization of different acyl-CoA substrates fo r acylation of lysoPC was different from that in microsomes, These res ults show that an enzyme specific for the formation of PC from lysoPC can be isolated in PC vesicles with a designed phospholipid molecular species composition and that the lipid environment plays sin important role in the regulation of the enzyme's affinity for its substrates.