Substrate specificity of acyl-CoA:Lysophospholipid acyltransferase (LAT) from pig spleen.

The present investigation was undertaken to gain insights into the nature of both substrate binding sites of acyl-CoA:lysophospholipid acyltransferase (LAT) which could be potentially useful for the identification and purification of this specific acyltransferase. Therefore, we have investigated the specificity of LAT from crude membranes of pig spleen toward various 1-palmitoyl-glycerophospholipids and 1-acyl-glycerophosphocholines (1-acyl-GPC). The enzyme showed the highest specificity toward 1-acyl-GPC and was able to distinguish between the acyl-chain length of the 1-acyl group within the 1-acyl-GPC molecule. We found preferential reactivity in the order C10:0 < C12:0 < C14:0, C18:0, C16:0 < C18:1 of 1-acyl-GPC. Lysophosphatidic acid or 1-O-alkyl-GPC were only poor substrates for the enzyme. In competition studies we could show that palmitic acid, oleic acid, arachidonic acid, and palmitoyl-CoA competitively inhibited LAT activity, whereas the coenzyme A failed to inhibit LAT enzyme activity in a concentration-dependent manner. We concluded that the ligand acyl-CoA is bound via its acyl chain. The finding that palmitoyl-CoA was a poor substrate as well as an inhibitor was the basis for protein purification. When palmitoyl-CoA-agarose was used as matrix for affinity chromatography, LAT enzyme activity was bound and eluted by high salt concentrations yielding an estimated 10-fold purification of the solubilized LAT enzyme.