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，已被证明在动物细胞中作为几种同工酶存在。已成功克隆了大鼠肝酶主要来源酵母酶和人胶质母细胞瘤酶的cDNA。大鼠肾酶已被纯化到均匀性，其分子大小（42 kDa）与大鼠肝脏或人胶质母细胞瘤酶相比，显示出相当小，据报道，它们分别为50 kDa或50 kDa。在本项目中，我们首先纯化了42 kDa的胆碱（乙醇胺）激酶（CK，EK），并通过胰蛋白酶酶切得到了该酶衍生的几个肽的部分氨基酸（AA）序列。由此得到的AA序列不仅与其他CK （EK）同工酶的AA序列不相同，而且与其他任何蛋白质的AA序列也不相同。然后，我们制备了几种混合寡核苷酸探针来筛选在lambda gt 10上构建的大鼠肾脏cDNA文库。到目前为止，已经分离出几个阳性克隆，并确定了插入的cdna的核苷酸序列。然而，这些克隆中的任何一个都没有显示出精确的大鼠肾42 kDa CK （EK）序列。寻找肾型CK cDNA仍在研究中。

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.