醛糖还原酶抑制剂筛选模型的建立
Setup model of screening aldose reductase inhibitor
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摘要: 目的: 建立醛糖还原酶抑制剂筛选模型,提供筛选醛糖还原酶抑制剂的方法。方法: 从大鼠晶状体中提取醛糖还原酶,设定10 mmol/L DL-甘油醛为底物,0.15 mmol/L NADPH为辅酶等条件建立初始酶促反应体系,维持其它条件不变改变底物浓度、辅酶浓度、反应温度和反应时间,选择酶活性峰值点建立模型,通过比较槲皮素与芦丁对醛糖还原酶的抑制作用检验此模型。结果: 醛糖还原酶酶促反应体系组成: 总体积1 ml,各反应液的终浓度分别为: DL-甘油醛10 mmol/L,NADPH0.15 mmol/L,缓冲液PBS的pH=6.2,120 mmol/L,醛糖还原酶提取物100μl,反应温度37℃,反应时间3 min。IR50槲皮素<IR50芦丁,槲皮素对大鼠晶状体醛糖还原酶的抑制作用强于芦丁。结论: 建立的醛糖还原酶抑制剂模型合理,易于操作,为筛选醛糖还原酶抑制剂提供了方法学基础。Abstract: Objective: To set up model of screening aldose reductase inhibitor.Methods: Aldose reductase was purified from rat lens.First,the activity of aldose reductase was assayed spectrophotometrically by following decrease in the absorption at 340nm over 3 min period with DL-glyceraldehydethe,10 mmol/L as substrate and NADPH,0.15 mmol/L as coenzyme.Then,we changed the condition respectively and independently.Last,the inhibition of aldose reductase from rat lens was compared by quercetin with rutoside by this model.Results: Each 1 ml cuvette contained: DL-glyceraldehydethe,10 mmol/L as substrate and NADPH,0.15mmol/L as coenzyme,the extract of aldose reductase,100 μl.The activity of aldose reductase was assayed at 340nm over 3 min period at 37 ℃.The inhibition of aldose reductase from rat lens by quercetin was better than rutoside from IR50 quercetin ＜ IR50 rutoside.Conclusion: This model is reasonable and convenient.
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