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目的从鸡肝中提取3-脱氧葡萄糖醛酮(3-DG)还原酶,并建立3-DG还原酶体外反应体系筛选药物。方法以辅酶NADPH的消耗量为评价指标来表示3-DG还原酶的活性,通过对反应体系底物3-DG、辅酶NADPH、酶量和反应时间的优化,考察温度等影响因素,建立3-DG还原酶体外反应体系并应用于寻找提高3-DG还原酶活性的药物。结果优化后的反应体系为:100μg.mL-1 3-DG溶液、250μg.mL-1 NADPH、酶液各20μL,用50 mmol.L-1的Na2HPO4-NaH2PO4缓冲液(pH6.4)调反应体系至200μL,混合物于37℃反应40 min;以NADPH的消耗量来定义3-DG还原酶的酶活单位,并应用于17种药物的筛选。结论该反应体系可用辅酶NADPH的消耗来反映体系中3-DG还原酶的活性,并能为寻找提高3-DG代谢酶活性的药物提供方法。
Objective To extract 3-deoxyglucosone (3-DG) reductase from chicken liver and establish a 3-DG reductase in vitro reaction system to screen the drug. Methods The activity of 3-DG reductase was expressed by the consumption of coenzyme NADPH. The effects of temperature and other factors were investigated by optimizing the substrate 3-DG, coenzyme NADPH, enzyme amount and reaction time, DG reductase in vitro reaction system and applied to find a drug that enhances 3-DG reductase activity. Results The optimized reaction system was as follows: 100μg.mL-1 3-DG solution, 250μg.mL-1 NADPH, 20μL enzyme solution and 50mmol.L-1 Na2HPO4-NaH2PO4 buffer (pH6.4) System to 200 μL, and the mixture was reacted at 37 ° C for 40 min. The enzyme activity unit of 3-DG reductase was defined by the consumption of NADPH and applied to the screening of 17 drugs. Conclusion The reaction system can use the consumption of coenzyme NADPH to reflect the activity of 3-DG reductase in the system, and can provide a method for finding a drug that enhances the activity of 3-DG metabolizing enzyme.