Shen Jie, Chen Wendong, Ji Kaida, Gao Pingjin, Zhu Dingliang
Department of Cardiology, Children's Hospital of Zhejiang University School of Medicine, Hangzhou 310051, China.
Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2017 May 25;46(6):643-648. doi: 10.3785/j.issn.1008-9292.2017.12.11.
To verify whether the enzymatic activity of kynureninase (KYNU) could be changed by the Arg188Gln (G/A) mutation. The total RNA of human hepatic tissue was extracted and the gene cDNA was amplified by RT-PCR. Primers were designed according to the sequences around the site Arg188Gln of gene and the Arg188Gln (G/A) mutant cDNA was generated by site-directed mutagenesis. Both the wild-type and mutant-type genes were subcloned into pcDNA vectors and the recombinant plasmids were constructed. After being transfected into human embryonic kidney 293 (HEK293) cells, the expression of KYNU recombinant plasmids were assessed by Western blot. The enzymatic activities of KYNU were detected by high performance liquid chromatography (HPLC). The KYNU enzyme activities were expressed in both wild and mutant HEK293 cells. Michaelis constants (Km) of the wild and mutant KYNU were (9.833±0.513) μmol/L and (29.900±0.265) μmol/L, respectively (<0.05). The maximum velocities (Vmax) of the wild and mutant KYNU were (0.700±0.096) nmol·mg·min and (0.084±0.003) nmol·mg·min, respectively (<0.05). Arg188Gln (G/A) mutation can decrease the enzymatic activity of KYNU.
为验证犬尿氨酸酶（KYNU）的酶活性是否会因Arg188Gln（G/A）突变而改变。提取人肝组织的总RNA，并通过RT-PCR扩增基因cDNA。根据基因Arg188Gln位点周围的序列设计引物，并通过定点诱变产生Arg188Gln（G/A）突变体cDNA。将野生型和突变型基因均亚克隆到pcDNA载体中，构建重组质粒。转染到人胚肾293（HEK293）细胞后，通过蛋白质免疫印迹法评估KYNU重组质粒的表达。通过高效液相色谱法（HPLC）检测KYNU的酶活性。野生型和突变型HEK293细胞中均表达KYNU酶活性。野生型和突变型KYNU的米氏常数（Km）分别为（9.833±0.513）μmol/L和（29.900±0.265）μmol/L（P<0.05）。野生型和突变型KYNU的最大反应速度（Vmax）分别为（0.700±0.096）nmol·mg·min和（0.084±0.003）nmol·mg·min（P<0.05）。Arg188Gln（G/A）突变可降低KYNU的酶活性。
