【摘要】 目的旨在了解人UGT1A3重组酶与芹菜素的有关代谢及其酶动力学参数,并阐述不同有机溶剂对酶动力学参数测定的影响。方法采用Bac-to-Bac系统表达人UGT1A3重组酶与芹菜素37℃共孵育,用HPLC法测定孵育液中剩余底物浓度,利用Lineweaver Burk法计算酶动力学参数,并进一步进行代谢物的确认。同时考察了用不同有机溶剂溶解底物对酶动力学参数测定的影响。结果采用Bac-to-Bac系统表达人UGT1A3重组酶,测得其催化芹菜素的Km为(28.88±2.47)μmol.L-1,Vmax为(224.19±21.11)nmol.m in-1.g-1,Vmax/Km为(7.75±0.29)mL.m in-1.g-1。不同有机溶剂对酶动力学参数测定无显著影响。结论芹菜素可被人UGT1A3重组酶催化,进行葡醛酸结合反应。更多还原

【Abstract】 AIM In order to explore the relationship between human UGT1A3 and apigenin metabolism,its kinetic parameters,and the effects of organic solvents.METHODS The Bac-to-Bac expression system was used to express the recombinant UGT1A3.The enzyme activity towards apigenin in cell homogenate protein was measured by HPLC.The kinetic parameters effected by organic solvents were also measured by the same methods.The metabolite was validated by β-glucuronidase and mass chromatogram.RESULTS UGT1A3 activity towards apigenin in cell homogenate protein was found to be K_m（28.88±2.47）μmol·L^-1,V_max（224.19±21.11）nmol·min^-1·g^-1,V_max/K_m（7.75±0.29）mL·min^-1·g^-1（n=3）.The organic solvents generally had a relatively minor effect on the enzyme activity.CONCLUSION（UGT1A3 catalyzed the） glucuronidation of apigenin,and organic solvents have a minor affect on UGT1A3 activity.更多还原