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浙江大学学报（医学版）
浙江大学学报（医学版）  2017, Vol. 46 Issue (6): 643-648    DOI: 10.3785/j.issn.1008-9292.2017.12.11
原著     
Arg188Gln(G/A)突变对犬尿氨酸酶活性的影响
沈杰1,*(),陈闻东2,姬开达2,高平进2,朱鼎良2
1. 浙江大学医学院附属儿童医院滨江院区心血管内科, 浙江 杭州 310051
2. 上海交通大学医学院附属瑞金医院上海市高血压研究所, 上海 200025
Effect of Arg188Gln (G/A) mutation on enzymatic activity of kynureninase
SHEN Jie1,*(),CHEN Wendong2,JI Kaida2,GAO Pingjin2,ZHU Dingliang2
1. Department of Cardiology, Children's Hospital of Zhejiang University School of Medicine, Hangzhou 310051, China
2. Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
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摘要：

目的: 验证犬尿氨酸酶（KYNU）Arg188Gln（G/A）突变能否改变KYNU的活性。方法: 抽提人肝脏细胞的总RNA，通过RT-PCR法获得KYNU基因全长cDNA，以KYNU基因Arg188Gln位点附近序列为模板设计定点突变引物，完成Arg188Gln（G/A）的定点突变，将突变型和野生型KYNU基因克隆到pcDNA载体质粒中，获得野生型和突变型pcDNA-KYNU重组表达质粒，经酶切鉴定和测序验证后转染HEK293细胞，蛋白质印迹法检测KYNU在细胞中的表达，用高效液相色谱法检测HEK293细胞的KYNU活性。结果: 野生型和突变型pcDNA-KYNU重组质粒在HEK293细胞中均能表达有活性的KYNU，KYNU的米氏常数分别为（9.833±0.513）μmol/L和（29.900±0.265）μmol/L，最大酶促反应速率分别为（0.700±0.096）nmol·mg-1·min-1和（0.084±0.003）nmol·mg-1·min-1，差异均有统计学意义（均P < 0.05）。结论: Arg188Gln（G/A）突变可以减弱KYNU的活性。
关键词： 犬尿氨酸/分析;  犬尿氨酸3-单加氧酶/分析;  基因;  突变;  高血压/病因学;  载体蛋白质类    
Abstract:

Objective: To verify whether the enzymatic activity of kynureninase (KYNU) could be changed by the Arg188Gln (G/A) mutation. Methods: The total RNA of human hepatic tissue was extracted and the KYNU gene cDNA was amplified by RT-PCR. Primers were designed according to the sequences around the site Arg188Gln of KYNU gene and the Arg188Gln (G/A) mutant KYNU cDNA was generated by site-directed mutagenesis. Both the wild-type and mutant-type KYNU 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). Results: 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 (P < 0.05). The maximum velocities (Vmax) of the wild and mutant KYNU were (0.700±0.096) nmol·mg-1·min-1 and (0.084±0.003) nmol·mg-1·min-1, respectively (P < 0.05). Conclusion: Arg188Gln (G/A) mutation can decrease the enzymatic activity of KYNU.
Key words: Kynurenine/analysis    Kynurenine 3-monooxygenase/analysis    Genes    Mutation    Hypertension/etiology    Carrier proteins
收稿日期: 2017-06-01 出版日期: 2017-12-25
CLC:  R394.3  
基金资助: 国家自然科学基金(30270545);浙江省中医药科技计划(2009CB048)
通讯作者: 沈杰     E-mail: shenjie112@zju.edu.cn
作者简介: 沈杰(1973-), 女, 博士, 主治医师, 主要从事心血管疾病的研究; E-mail:shenjie112@zju.edu.cn; https://orcid.org/0000-0002-8539-2829
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引用本文:

沈杰,陈闻东,姬开达,高平进,朱鼎良. Arg188Gln(G/A)突变对犬尿氨酸酶活性的影响[J]. 浙江大学学报（医学版）, 2017, 46(6): 643-648.

SHEN Jie,CHEN Wendong,JI Kaida,GAO Pingjin,ZHU Dingliang. Effect of Arg188Gln (G/A) mutation on enzymatic activity of kynureninase. J Zhejiang Univ (Med Sci), 2017, 46(6): 643-648.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2017.12.11    或    http://www.zjujournals.com/med/CN/Y2017/V46/I6/643

图 1  pcDNA-KYNU重组质粒的电泳结果
图 2  蛋白质印迹法检测野生型和突变型HEK293细胞表达KYNU的结果
图 3  不同底物浓度对犬尿氨酸酶(KYNU)酶促反应速率的影响
($\bar x \pm s$)
HEK293细胞 米氏常数(μmol/L) 最大反应速率
(nmol·mg-1·min-1)
 野生型 9.833±0.513 0.700±0.096
 突变型 29.900±0.265 0.084±0.003
 t值 -60.2 11.143
 P值 < 0.05 < 0.05
表 1  野生型和突变型HEK293细胞表达的犬尿氨酸酶(KYNU)酶促反应动力学指标测定结果
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