Characterization of a lipid activated CTP:: phosphocholine cytidylyltransferase from Drosophila melanogaster
Characterization of a lipid activated CTP:: phosphocholine cytidylyltransferase from Drosophila melanogaster
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DOI：
10.1016/j.bbalip.2004.04.008
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发表时间：
2004-07-05
影响因子：
4.8
通讯作者：
Friesen, JA
中科院分区：
文献类型：
--
作者：
Helmink, BA;Friesen, JA
CTP：磷酸胆碱胞苷酰转移酶 (CCT) 是细胞磷脂酰胆碱 (PC) 合成的关键酶，可将磷酸胆碱和胞苷 5'-三磷酸 (CTP) 转化为 CDP-胆碱。我们从果蝇中分离出编码 CCT 亚型的 cDNA，并使用杆状病毒表达系统在草地贪夜蛾 (Sf9) 昆虫细胞中表达重组天然形式和 6 x -His 标记形式。使用抗磷酸氨基酸抗体的免疫印迹显示该酶在丝氨酸和苏氨酸残基上被磷酸化，但在酪氨酸上未被磷酸化。纯化的天然酶表现出1352+/-159nmol CDP-胆碱/分钟/mg的V-max值、磷酸胆碱的0.50+/-0.09mM的K值和CTP的0.72+/-0.10mM的K'(希尔常数)值。 6 X-His 标记的酶具有类似的特性，V-max 值为 2254 +/- 253 nmol CDP-胆碱/min/mg，磷酸胆碱的 K-m 值为 0.63 +/- 0.13 mM，CTP 的 K' 等于 0.81 +/- 0.20 mM。每种形式的酶被含有 50 mol% 油酸盐的合成 PC 囊泡激活到相似的程度。使用含有二磷脂酰甘油（DPG）的PC囊泡时，脂质激活效率最高，当磷脂酰丝氨酸（PS）和磷脂酰肌醇（PI）掺入囊泡中时，激活效率明显较低，单独的PC或含有磷脂酰乙醇胺的PC囊泡是效率最低的酶激活剂。 (C) 2004 Elsevier B.V. 保留所有权利。
CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5' -triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6 x -His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V-max value of 1352 +/- 159 nmol CDP-choline/min/mg, a K. value of 0.50 +/- 0.09 mM for phosphocholine, and a K' (Hill constant) value of 0.72 +/- 0.10 mM for CTP. The 6 X -His-tagged enzyme has similar properties with a V-max value of 2254 +/- 253 nmol CDP-choline/min/mg, a K-m value of 0.63 +/- 0.13 mM for phosphocholine and a K' for CTP equal to 0.81 +/- 0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators. (C) 2004 Elsevier B.V. All rights reserved.