Expression and characterization of the active molecular forms of choline/ethanolamine kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
Expression and characterization of the active molecular forms of choline/ethanolamine kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
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DOI：
10.1042/0264-6021:3630777
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发表时间：
2002-05-01
影响因子：
4.1
通讯作者：
Ishidate, K
中科院分区：
文献类型：
--
作者：
Aoyama, C;Ohtani, A;Ishidate, K
胆碱/乙醇胺激酶（ChoK/EtnK）在哺乳动物细胞中以至少三种同种型（α 1、α 2和β）存在。然而，这种酶存在一种以上形式的生理意义仍有待确定。在本研究中，我们研究了表达和分布的异构体在小鼠组织中使用异构体特异性的cDNA探针和多克隆抗体提出对每个N-末端肽序列。北方和西方印迹分析表明，无论是α（α 1加α 2）或β亚型似乎是普遍表达的酶。睾丸中α亚型的mRNA丰度最高，而心脏和肝脏中β亚型的mRNA丰度相对较高。虽然每种同种型的天然形式被报道由同源二聚体或同源四聚体组成，但我们的免疫滴定研究清楚地表明，相当大一部分酶的活性形式由α/β异源寡聚体组成，相对较小的活性部分由α/α和β/β同源寡聚体表达。这是第一个实验证据的存在异聚ChoK/ EtnK的来源。因此，我们的研究结果强烈表明，细胞中ChoK/EtnK的活性不仅受每种亚型的水平控制，而且还受它们组合形成活性寡聚体复合物的控制。四氯化碳（CCl 4）显示在小鼠肝脏中诱导ChoK活性2-4倍。我们对这种诱导机制的分析表明，CCl 4的负责亚型是α，而不是β。在小鼠肝脏中强烈诱导α mRNA的水平，这导致α亚型的量持续增加。因此，α/α同源寡聚体的组成在CCl 4诱导的肝脏中占ChoK总活性分子形式的80%，而在正常未诱导的肝脏中不到20%。
Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homo-tetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/ EtnK in an source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl4) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl4 was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl4-induced liver, whereas it was less than 20% in normal uninduced liver.