PC and PE synthesis: Mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1)

Abstract The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual‐specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP‐choline or CDP‐ethanolamine to DAG to form PC and PE. We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1. A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides. The CEPT1 enzyme displayed an apparent K m of 36 μM for CDP‐choline and 4.2 mol% for di‐18∶1 DAG with a V max of 14.3 nmol min −1 mg −1 . When CDP‐ethanolamine was used as substrate, the apparent K m was 98 μM for CDP‐ethanolamine and 4.3 mol% for di‐18∶1 DAG with a V max of 8.2 nmol min −1 mg −1 . The preferred diradylglycerol substrates used by CEPT1 with CDP‐choline as the phosphobase donor were di‐18∶1 DAG, di‐16∶1 DAG, and 16∶0/18∶1 DAG. A major difference between previous emulsion‐based assay results and the mixed micelle results was a complete inability to use 16∶0(O)/2∶0 as a substrate for the de novo synthesis of platelet‐activating factor when the mixed micelle assay was used. When CDP‐ethanolamine was used as the phosphobase donor, 16∶0/18∶1 DAG, di‐18∶1 DAG, and di‐16∶1 DAG were the preferred substrates. The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1. The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC 50 values of 40 μM.