Cloning and characterization of a lipid-activated CTP:phosphocholine cytidylyltransferase from Caenorhabditis elegans: identification of a 21-residue segment critical for lipid activation.
Cloning and characterization of a lipid-activated CTP:phosphocholine cytidylyltransferase from Caenorhabditis elegans: identification of a 21-residue segment critical for lipid activation.
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脂质激活的 CTP：来自秀丽隐杆线虫的磷酸胆碱胞苷酰转移酶的克隆和表征：鉴定对脂质激活至关重要的 21 个残基片段。
DOI：
10.1016/s1388-1981(01)00145-7
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Kent,C
中科院分区：
文献类型：
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作者：
Friesen,JA;Liu,MF;Kent,C
秀丽隐杆线虫的基因组包含几个基因，似乎编码类似于CTP的蛋白质：磷酸胆碱胞苷酰转移酶（CCT）。我们从一株C.从线虫cDNA文库中获得了编码与先前鉴定的CCT最相似的CCT的347个氨基酸版本的cDNA文库。使用杆状病毒表达系统表达和纯化天然和His标记形式。5 μM磷脂酰胆碱：油酸酯（50：50）囊泡可最大程度地激活该酶，在存在脂质的情况下，其kcat值是不存在脂质的情况下的kcat值的37倍。定位C.在对脂质活化至关重要的线虫CCT中，分析了一系列C-末端截短突变体。在氨基酸225或245之后截短的CCT在不存在脂质的情况下是相当有活性的，并且在存在脂质的情况下不进一步活化，这支持了脂质活化区段在不存在脂质的情况下抑制催化的概念。氨基酸266、281或319后截短的CCT被脂质激活，类似于野生型酶。在不存在脂质的情况下的动力学分析揭示了在氨基酸245之后截短的脂质非依赖性CCT具有比全长CCT或在氨基酸266之后截短的CCT大15倍的kcat值。我们的结论是激活C.通过脂质引起的秀丽线虫CCT包含在氨基酸246-266内，该区域在不存在脂质的情况下是抑制性的，并且该抑制通过酶与脂质的缔合而减轻。
The genome of the nematode Caenorhabditis elegans contains several genes that appear to encode proteins similar to CTP:phosphocholine cytidylyltransferase (CCT). We have isolated a 1044-nucleotide cDNA clone from a C. elegans cDNA library that encodes the 347-amino acid version of CCT that is most similar to previously-identified CCTs. Native and His-tagged forms were expressed and purified using a baculovirus expression system. The enzyme was maximally activated by 5 μM phosphatidylcholine:oleate (50:50) vesicles with a kcatvalue in the presence of lipid 37-fold greater than the kcatvalue in the absence of lipid. To localize the region of C. elegans CCT critical for lipid activation, a series of C-terminal truncation mutants was analyzed. CCT truncated after amino acids 225 or 245 was quite active in the absence of lipids and not further activated in the presence of lipids, supporting the concept that the lipid-activation segment is inhibitory to catalysis in the absence of lipids. CCT truncated after amino acids 266, 281, or 319 was activated by lipid similar to wild-type enzyme. Kinetic analysis in the absence of lipid revealed the lipid-independent CCT truncated after amino acid 245 to have a kcatvalue 15-fold greater than either full-length CCT or CCT truncated after amino acid 266. We conclude that elements critical for activation of C. elegans CCT by lipids are contained within amino acids 246–266, that this region is inhibitory in the absence of lipids, and that the inhibition is relieved by the association of the enzyme with lipid.