Wright Marcia M, McMaster Christopher R
Atlantic Research Centre, Department of Pediatrics, IWK Health Centre, Dalhousie University, Halifax, Nova Scotia, Canada.
Lipids. 2002 Jul;37(7):663-72. doi: 10.1007/s11745-002-0947-6.
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 Km of 36 microM for CDP-choline and 4.2 mol% for di-18:1 DAG with a Vmax of 14.3 nmol min(-1) mg(-1). When CDP-ethanolamine was used as substrate, the apparent Km was 98 microM for CDP-ethanolamine and 4.3 mol% for di-18:1 DAG with a Vmax 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 IC50 values of 40 microM.
人类胆碱/乙醇胺磷酸转移酶1（CEPT1）基因编码一种双特异性酶，该酶通过将磷酸碱基从CDP - 胆碱或CDP - 乙醇胺转移至二酰甘油（DAG）以形成磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE），从而催化这两种主要磷脂的从头合成。我们使用了一种缺乏内源性胆碱磷酸转移酶和乙醇胺磷酸转移酶活性的表达系统来评估CEPT1对二酰甘油的特异性。采用混合胶束测定法以确保所递送的二酰甘油不会影响CEPT1所处的膜环境。CEPT1酶对CDP - 胆碱的表观Km为36μM，对二 - 18:1 DAG为4.2 mol%，Vmax为14.3 nmol min⁻¹ mg⁻¹。当使用CDP - 乙醇胺作为底物时，对CDP - 乙醇胺的表观Km为98μM，对二 - 18:1 DAG为4.3 mol%，Vmax为8.2 nmol min⁻¹ mg⁻¹。以CDP - 胆碱作为磷酸碱基供体时，CEPT1偏好使用的二酰甘油底物为二 - 18:1 DAG、二 - 16:1 DAG和16:0/18:1 DAG。先前基于乳液的测定结果与混合胶束结果之间的一个主要差异在于，当使用混合胶束测定法时，完全无法将16:0(O)/2:0用作从头合成血小板活化因子的底物。当使用CDP - 乙醇胺作为磷酸碱基供体时，16:0/18:1 DAG、二 - 18:1 DAG和二 - 16:1 DAG是偏好的底物。混合胶束测定法还可用于测量CEPT的脂质活化，胆碱磷酸转移酶和乙醇胺磷酸转移酶活性均在5 mol%时表现出产物活化的异常特性，这意味着CEPT1上存在特定的脂质活化结合位点。蛋白激酶C抑制剂白屈菜红碱和人二酰甘油激酶抑制剂R59949均抑制CEPT1活性，IC50值为40μM。
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