MES23.5细胞酪氨酸羟化酶的种属来源及其重组酶的活性测定

MES 2 3 5细胞作为研究神经变性疾病的工具 ,是一种杂交瘤性多巴胺能神经元细胞系 ,由大鼠胚胎中脑细胞与小鼠神经母细胞瘤 胶质瘤细胞系N18TG2杂交而成 .其酪氨酸羟化酶 (tyrosinehydroxylase ,TH)的动物种属来源不清楚 .应用反转录聚合酶链反应 (RT PCR)法从MES 2 3 5细胞系中克隆了编码TH的cDNA ;结构分析表明 ,其cDNA编码区由 14 97碱基构成 ,共编码 4 98个氨基酸 ,与大鼠和小鼠TH的同源程度分别为 93%和 10 0 % .该杂交瘤细胞系表达小鼠TH .将该cDNA亚克隆至原核表达载体pGEX 4T 1,经原核细胞表达、亲和层析得到了电泳纯的基因重组小鼠TH(recombinantmousetyrosinehydroxylase ,rmTH) .改良和建立了一种体外分析TH活性的新方法 .活性分析证明 ,纯化的rmTH能催化L 酪氨酸发生加单氧反应生成L 3,4 二羟基苯丙氨酸 (L 多巴 ) .rmTH的表观分子量为 5 6kD ;其酶促加单氧反应的最适pH值为 7 0 .乙二胺四乙酸能显著抑制此酶的活性 ,而亚铁离子能明显增强其活性 .

Identification and Activity Assay of the Tyrosine Hydroxylase in MES23.5 Cells

MES23.5 cell line, which served as a useful tool for studying neurodegenerative disease, is a dopaminergic neuroblastoma derived from somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cells. But the origin of its tyrosine hydroxylase (TH) was unclear. A TH cDNA was cloned from MES23.5 cells by RT-PCR. The 1497 bp cDNA, encoding 498 amino acids, is completely identical to the mouse TH and shares 93% homology with rat TH. These results reveal that MES cells express mouse TH. The coding region of the cDNA was subcloned into a bacteria expression vector pGEX-4T-1, and the resulting TH-GST fusion protein was expressed at a high level in E. coli and purified via affinity chromatography. An improved method is used to analyze the activity of TH in vitro. The specific enzyme activity was 0.2698 μmol/min per mg of protein. The molecular mass of rTH—? ?6 000 was measured by SDS-polyacrylamide gel electrophoresis and the optimal pH of the enzymatic activity was 7.0. The activity was significantly inhibited by EDTA, and was markedly activated by Fe(Ⅱ).