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To analyse the involvement of the plastidial lysophosphatidylcholine (lyso-PC) acyltransferase in the import of the extraplastidial lipid precursors required for eukaryotic plastid lipid synthesis, we plan to obtain transgenic plants. Since no sequence of lyso-PC acyltransferase is known, the purification of this enzyme has been undertaken to establish its sequence. First we determined the conditions allowing the solubilization of this membrane-bound enzyme. It is shown that by using CHAPS as a detergent, a lyso-PC acyltransferase activity is associated with the solubilized proteins.

Understanding the three-dimensional structure of G protein-coupled receptors (GPCRs) has been limited by the technical challenges associated with expression, purification, and crystallization of membrane proteins, and their low abundance in native tissue. In the first large-scale comparative study of GPCR protein production using recombinant baculovirus, we report the characterization of 16 human receptors. The GPCRs were produced in three insect cell lines and functional protein levels monitored over 72 h using radioligand binding assays. Different GPCRs exhibited widely different expression levels, ranging from less than 1 pmol receptor/mg protein to more than 250 pmol/mg. No single set of conditions was suitable for all GPCRs, and large differences were seen for the expression of individual GPCRs in different cell lines. Closely related GPCRs did not share similar expression profiles; however, high expression (greater than 20 pmol/mg) was achieved for over half the GPCRs in our study. Overall, the levels of protein production compared favourably to other published systems.

Imidazole catalyzed acylations of lysolipids by acyl-CoAs in water at room temperature and at a pH close to neutrality. In the presence of oleoyl-CoA and either lysophosphatidylcholine, 1-palmitoyl-sn-glycero-3-phosphocholine (LPC); lysophosphatidylglycerol, monoacyl-sn-glycero-3-phosphoglycerol; lysophosphatidyl acid, 1-oleoyl-sn-glycero-3-phosphate; lysophosphatidylserine, monoacyl-sn-glycero-3-phosphoserin; or lysophosphatidylethanolamine, monoacyl-sn-glycero-3-phosphoethanolamine, the corresponding phospholipids were synthesized. Similarly, the use of lyso-platelet activating factor, an ether analog of LPC, yielded the formation of 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine. In the presence of LPC, an imidazole-catalyzed synthesis of phosphatidylcholine (PC) occurred when medium, long, and very long chain acyl-CoAs were added. With hydroxyacyl-CoA, a similar PC synthesis was obtained. The process described in the present paper appears to offer several potential applications of interest for the synthesis of glycerophospholipids and triglycerides with labeled and/or an unusual or fragile fatty acid, or when suitable acyltransferases have not yet been described in the literature and/or are not commercially available. The method described is very safe and simple since lipids can be synthesized in tubes containing 0.7% imidazole in water, and left for a few hours at room temperature on the bench.