Regulation of choline (ethanolamine) kinase gene expression

胆碱(乙醇胺)激酶是磷脂酰胆碱(磷脂酰乙醇胺)生物合成的关键酶，在动物细胞中以几种同工酶的形式存在。其主要来源酵母酶和人脑胶质母细胞瘤酶均已克隆成功。大鼠肾脏酶已得到纯化，其分子大小(42 KDa)与大鼠肝脏或人胶质母细胞瘤酶相比相当小，已有报道分别为50 kDa和50 kDa。在本项目中，我们首先纯化了42 kDa胆碱(乙醇胺)激酶(CK，EK)，并通过胰酶消化获得了几种多肽的部分氨基酸序列。由此获得的氨基酸序列不仅与已报道的其他CK(EK)同工酶的氨基酸序列不一致，而且与任何其他蛋白质的AA序列也不相同。在此基础上，我们制备了多种寡核苷酸混合探针，用于从大鼠肾脏中筛选已构建在lambda GT10上的cDNA文库，目前已分离到几个阳性克隆，并测定了插入的cDNAs的核苷酸序列。然而，这些克隆中的任何一个都没有被证明具有准确的大鼠肾脏42 kDa CK(EK)序列。对肾型CK基因的研究仍在进行中。

Choline (ethanolamine) kinase, a key enzyme of phosphatidylcholine (phosphatidylethanolamine) biosynthesis, has been shown to exist in animal cells as several isozymes. Its cDNA cloning has been done for the yeast enzyme, the major from of rat liver enzyme as well as for the human glioblastoma enzyme. Rat kidney enzyme has been purified to homogeneity and its molecular size (42 kDa) is shown to be rather small when compared to either rat liver or human glioblastoma enzyme, which has been reported to have 50 kDa or 50 kDa size, respectively. In this project, we first purified 42 kDa choline (ethanolamine) kinase (CK,EK) and obtained the partial amino acid (AA) sequences of several peptides derived from the purified enzyme by trypsin digestion. Any of the AA sequences thus obtained were not identical to the reported AA sequences of not only other CK (EK) isozymes but also any other proteins. Then, we prepared several mixed oligonucleotide probes to screen the cDNA library from rat kidney, which had been constructed on lambda gt 10. By now, several positive clones have been isolated and the nucleotide sequence of inserted cDNAs has been determined. However, any of these clones have not been shown to have exact rat kidney 42 kDa CK (EK) sequences. The search for the kidney type CK cDNA is still under investigation.