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J Zhejiang Univ (Med Sci)  2017, Vol. 46 Issue (6): 643-648    DOI: 10.3785/j.issn.1008-9292.2017.12.11
    
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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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 wordsKynurenine/analysis      Kynurenine 3-monooxygenase/analysis      Genes      Mutation      Hypertension/etiology      Carrier proteins     
Received: 01 June 2017      Published: 25 December 2017
CLC:  R394.3  
  R34  
Corresponding Authors: SHEN Jie     E-mail: shenjie112@zju.edu.cn
Cite this article:

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.

URL:

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


Arg188Gln(G/A)突变对犬尿氨酸酶活性的影响

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 
Fig 1 Electrophoretic analysis for pcDNA-KYNU recombination plasmid
Fig 2 Western blot result of wild and mutant KYNU expressed in HEK293 cells
Fig 3 Effects of different concentrations of substrate on KYNU enzymatic reaction velocity
($\bar x \pm s$)
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 -60.2 11.143
 P < 0.05 < 0.05
Tab 1 Kinetic properties of wild and mutant KYNU expressed in HEK293 cells
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