Roberti R, Vecchini A, Freysz L, Masoom M, Binaglia L
Department of Biochemistry, Faculty of Medicine, University of Perugia, Italy.
Biochim Biophys Acta. 1989 Jul 17;1004(1):80-8. doi: 10.1016/0005-2760(89)90216-6.
Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminephosphotransferase was strongly modified by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.
乙醇胺磷酸转移酶（CDP乙醇胺：1,2-二酰基甘油乙醇胺磷酸转移酶，EC 2.7.8.1）已通过两步色谱法从大鼠脑微粒体中以活性形式纯化出来。通过在含有1% Triton X-100的溶解缓冲液和洗脱缓冲液中添加0.01% 2,6-二叔丁基-4-甲基苯酚，获得了具有高比活性和稳定性的酶制剂。纯化酶的比活性比粗溶解酶高约1200倍。在存在Triton X-100的情况下以及在无去污剂的酶制剂中，均研究了乙醇胺磷酸转移酶的脂质依赖性。去污剂溶解的乙醇胺磷酸转移酶的活性受到磷脂的强烈影响。该酶的动力学行为还取决于从去污剂/脂质/蛋白质悬浮液中去除去污剂后得到的聚集体中所含的脂质。讨论了磷脂对膜结合乙醇胺磷酸转移酶活性的调节作用。
