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化学学报
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化学学报 ›› 2001, Vol. 59 ›› Issue (5): 788-792. 上一篇    下一篇

研究论文

哺乳动物细胞是所得多肽复合酶CAD催化反应产物的 HPLC分析

王 来来;胡霄雪;辛嘉英;崔俊儒;杨树本;朱利民   

  1. 中国科学院兰州化学物理研究所.兰州(730000);中国科学院羰基合成与选择国 家重点实验室;英国北伦敦大学生物和应用科学系
  • 发布日期:2001-05-15

HPLCseparation of the reaction mixture in mammalian cell catalyzed by the multienzyme

Wang lailai;Hu Xiaoxue;Xin Jiaying;Cui Junru;Yang Shuben;Zhu Limin   

  1. Lanzhou Inst Chem Phys, CAS.Lanzhou(730000)
  • Published:2001-05-15

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本研究报道了以150mm×4.6mmI.D.Zorbaxoqcyuosyg.10mmoI·L^-1KH~2PO~4和500mmoI·L^-1LKH~2PO~4水溶液为洗脱液，梯度程序为0～5min(φ=1，体积分数)10mmoI·L^-1KH~2PO~4溶液，5～50min由(φ=1)10mmoI·L^-1KH~2PO~4溶液转换到(φ=1)500mmol·L^-1KH~2PO~4溶液时，高效液相色谱分离了哺乳动物细胞中所得肽复合酶ACD催化反应混合物三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和二氢乳清酸(DHO)，确定了ATP，ADP和DHO的出峰顺序，制作了工作曲线，确定了催化反应混合物中ATP,ADP和DHO的相应浓度。

关键词: 三磷酸腺苷, 二磷酸腺苷, 高速液体色谱, 多肽, 复合酶, 二氢乳清酸

The product of the first three steps of de novo pathway of pyremidine biosynthesis in mammalian cell catalyzed by carbamoyl phosphate synthetase- asparate transcarbamoylase - dihyroorotase are adenosine diphosphate (ADP) and dihydroorotate (DHO. ). And one of reactant is adenosine tiphosphate(ATP). A HPLC method for separation of ADP, ATP and DHO was developed. A Zarbox column (150mm×4.6mmI.D.) was used. The mobile phase were 0.01mol·L^-1 potassium dihydrogen phosphate buffer(pH4.7) and 0.5mol·L^-1potassium dihydrogen phosphate (pH4.6). The gradient was programmed at 0～5min, (φ=1, volume ratio)0.01mol ·L^-1KH~2PO~4 then 5～50 min, (φ=1,volume ratio) 0.01molL^- 1KH~2PO~4 to (φ=1,volume ratio) 0.5molL^-1KH~2PO~4. The flow rate was 0.7 mL·min^-1. The optimum deteation wavelenght and the sequence of ATP, ADP and DHO peaks were detected. The working cursor was plotted and then the corresponding concentration of ATP, ADP and DHO in the catalytic reaction were calculated.

Key words: ADENOSINE TRIPHOSPHATE (ATP), HIGH SPEED LIQUID CHROMATOGRAPHY, POLYPEPTIDE

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