醛糖还原酶抑制剂筛选模型的建立和初步应用

Study on the primary screening model for the aldose reductase inhibitors

本研究从牛晶状体中提取醛糖还原酶（AR），以DL-甘油醛为底物、还原型辅酶Ⅱ（NADPH）为辅酶，应用96孔石英板建立了醛糖还原酶抑制剂（ARI）微量高效筛选模型。酶促反应体系体积为200μ1，其组成成分为：50mmol/L磷酸盐缓冲液（pH6．2）、400mmol／L硫酸锂、5mmol／L 2-巯基乙醇、0． 24mmol／L NADPH、1、2mg粗酶、2mmol/L DL-甘油醛，用蒸馏水补足体积。反应温度为37°C，规定反应体系吸光度每分钟下降0．001为一个酶活力单位。以不含底物的样品为空白对照。开展了从微生物代谢产物中筛选ARI的工作，已经累计筛选真菌和稀有放线菌发酵样品约1500个，其中获得阳性样品6个，阳性率约为0．4％。

ABSTRACT:Aldose reductase (AR) is a rate limiting enzyme in the polyol pathway with the conversion ofglucose to sorbitol. Experiments in diabetic animals and clinical trials implicate that AR is a potential target for theprevention and treatment of diabetic complications such as cataracts, retinopathy, nephropathy and neuroapthy.After homogenization, centrifugation, (NH4)2SO4 fractionation (0～75% ) and dialysis, the crude AR from calflenses was obtained. A primary screening method has been set up and AR activity at 37°C was photometricallydetermined by the decrease in absorbance at 340nm using DL-glyceraldehyde as substrate. During the research of newaldose reductase inhibitors (ARI), 6 samples from about 1500 microbial fermentation were screened. The positive rateis about 0. 4%.