龙葵碱对HepG_2人肝癌细胞NAT酶动力学常数的影响

目的探讨龙葵碱对HepG2细胞NAT酶米氏常数Km及最大反应速率Vmax的影响。方法MTT法测定龙葵碱对消化系统SGC-7901人胃癌、HepG2人肝癌、Ls-174人大肠癌3种肿瘤细胞株的细胞毒作用,采用高效液相色谱(HPLC)法,以2-AF为底物,以2-AF的浓度为底物浓度,在以HepG2完整细胞及细胞质内2-AF被NAT酶乙酰化为2-AAF的速度为NAT酶的反应速率,采用双倒数作图法,以底物2-AF浓度的倒数1/S对NAT反应速率的倒数1/V作直线,得出回归方程,计算Km和Vmax。结果MTT法测定表明龙葵碱对HepG2人肝癌细胞比较敏感,酶动力学研究表明,以2-AF为底物,对于HepG2完整细胞,阴性对照组的Km和Vmax分别为(2.37×10-3±8.37×10-5)mmol.L-1、(9.16×10-4±7.54×10-5)nmol.106cells-1,龙葵碱组的Km和Vmax分别为(2.22×10-3±9.05×10-5)mmol.L-1和(5.14×10-4±3.72×10-5)nmol.106cells-1。对于HepG2细胞质,阴性对照组的Km和Vmax分别为(8.95×10-3±2.61×10-4)mmol.L-1、(2.55×10-6±1.92×10-8)μmol.min-1g-1Pro,龙葵碱组的Km和Vmax分别为(9.48×10-3±3.63×10-4)mmol.L-1和(2.43×10-6±1.32×10-8)μmol.min-1g-1Pro,统计学表明对于HepG2完整细胞和细胞质,阴性对照组和龙葵碱组的Km没有差异,而Vmax差异有显著性(完整细胞P<0.01,细胞质P<0.05)。结论龙葵碱是HepG2人肝癌细胞NAT酶2-AF底物的非竞争性抑制剂。

Effects of solanine on kinetic constants of NATase in HepG2 cells

Aim To explore the effects of solanine on Km and Vmax of NATase of HepG2.Methods MTT assay was adopted to determine the cytotoxicity to SGC-7901,HepG2,and Ls-174.Employing HPLC,using 2-AF as substrate,taking concentration of 2-AF as concentration of substrate,in intact HepG2 cells and their cytoplasm,taking the speed of 2-AF being acetylated to 2-AFF by NATase as the rate of NATase,using double reciprocal plot,taking 1/S(the reciprocal of concentration of 2-AF) and 1/V(reaction rate of NATase) as coordinates,regression equation was obtanined,and Km and Vmax were calculated.Results Solanine was cytotoxic to HepG2.Study on enzyme kinetics demonstrated,as for intact HepG2 cells,Km and Vmax of control group were(2.37×10-3±8.37×10-5) mmol·L-1,(9.16×10-4±7.54×10-5) nmol·(106 cells)-1 respectively,Km and Vmax of the solanine group were(2.22×10-3±9.05×10-5) mmol·L-1,(5.14×10-4±3.72×10-5)nmol·(106 cells)-1 respectively.As for the cytoplasm of HepG2 cells,Km and Vmax of control group were(8.95×10-3±2.61×10-4) mmol·L-1 and(2.55×10-6±1.92×10-8)μmol·min-1·g-1 protein,Km and Vmax of the solanine group were(9.48×10-3±3.63×10-4) mmol·L-1 and(2.43×10-6±1.32×10-8) μmol·min-1·mg-1 protein.Statistically,as for intact HepG2 cells and their cytoplasm,there was no difference between the Km of control group and that of solanine group,but there was remarkable difference between Vmax of control group and that of solanine group,P<0.01 for intact cell and P<0.05 for cytoplasm.Conclusion Solanine was a non-competitive inhibitor of NATs in HepG2.