Moon E A, O'Neill C
Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St Leonards, NSW, Australia.
J Reprod Fertil. 1997 Jul;110(2):213-8. doi: 10.1530/jrf.0.1100213.
Cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyses the synthesis of the active metabolic intermediate cytidine diphosphocholine (which is mainly used in the synthesis of choline-containing phospholipids). It is a rate-limiting reaction in choline phospholipid biosynthesis in many cells. In this study, a microassay is reported for the detection of this enzyme in small numbers of cells. This enzyme was present in mouse oocytes and at all stages during preimplantation development. Enzyme activity was destroyed by boiling but increased with time and number of embryos in the reaction. Activity in two-cell embryos was dependent on Mg2+ but independent of Ca2+ and was enhanced by the addition of 1 microgram lysophosphatidylethanolamine ml-1 to the reaction mixture. Activity was apparently dependent upon the phosphorylation status of the enzyme since the absence of the phosphatase inhibitor NaF caused a significant inhibition of activity. The enzyme in oocytes had a specific activity of 2.8 +/- 0.3 fmol cytidine diphosphocholine (CDP-choline) per oocyte min-1 (mean +/- SEM). The specific activity in two-cell and eight-cell embryos and blastocysts was not different from that of oocytes. Fertilized one-cell embryos had significantly less activity (1.4 +/- 0.05 fmol CDP-choline produced per embryo min-1) than other stages studied. Furthermore, the enzyme present in one-cell embryos was not capable of being further activated by the addition of exogenous lysophosphatidylethanolamine to the reaction. The increase in activity from the one-cell to the two-cell stage was not inhibited by alpha-amanitin (an inhibitor of RNA polymerase II), cycloheximide (a protein synthesis inhibitor) [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl]dihydrochloride (H-7; a protein kinase inhibitor) and was independent of cell-cycle progression; these results suggest that enzyme activity is independent of transcription, protein synthesis and the action of some kinases, including cell-cycle-dependent kinases. This study provides the first description of cytidylyltransferase in the early mammalian embryo.
胞苷5'-三磷酸（CTP）：磷酸胆碱胞苷转移酶（EC 2.7.7.15）催化活性代谢中间体胞苷二磷酸胆碱的合成（其主要用于含胆碱磷脂的合成）。在许多细胞中，这是胆碱磷脂生物合成中的限速反应。在本研究中，报道了一种用于检测少量细胞中该酶的微量测定法。该酶存在于小鼠卵母细胞以及植入前发育的各个阶段。酶活性经煮沸后被破坏，但在反应中会随时间和胚胎数量的增加而升高。二细胞胚胎中的活性依赖于Mg2+，但不依赖于Ca2+，并且向反应混合物中添加1微克溶血磷脂酰乙醇胺/毫升可增强活性。活性显然依赖于该酶的磷酸化状态，因为缺乏磷酸酶抑制剂氟化钠会导致活性显著抑制。卵母细胞中的该酶比活性为每个卵母细胞每分钟2.8±0.3飞摩尔胞苷二磷酸胆碱（CDP-胆碱）（平均值±标准误）。二细胞、八细胞胚胎和囊胚中的比活性与卵母细胞的比活性无差异。受精的一细胞胚胎的活性（每个胚胎每分钟产生1.4±0.05飞摩尔CDP-胆碱）明显低于所研究的其他阶段。此外，一细胞胚胎中存在的酶不能通过向反应中添加外源性溶血磷脂酰乙醇胺而进一步激活。从一细胞阶段到二细胞阶段活性的增加不受α-鹅膏蕈碱（RNA聚合酶II抑制剂）、环己酰亚胺（蛋白质合成抑制剂）[1-（5-异喹啉磺酰基）-2-甲基哌嗪，HCl]二盐酸盐（H-7；一种蛋白激酶抑制剂）的抑制，并且与细胞周期进程无关；这些结果表明酶活性独立于转录、蛋白质合成以及某些激酶的作用，包括细胞周期依赖性激酶。本研究首次描述了早期哺乳动物胚胎中的胞苷转移酶。
