人脑醛糖还原酶的纯化与性质

Purification and Properties of Aldose Reductase from Human Brain

联合采用ＤＥＡＥ－纤维素层析、色谱聚焦、ＮＡＤＰ亲和层析与ＳｅｐｈａｄｅｘＧ－１００的凝胶过滤，对人脑醛糖还原酶（ＥＣ１．１．１．２１；ＡＬＲ）进行纯化．现测得该酶的等电点ｐＨ值为５．８５．经聚丙烯酰胺凝胶盘状电泳和Ｗｅｓｔｅｒｎ印迹证实，获得了满意的酶纯度．同葡萄糖，葡糖－６－磷酸与ＮＡＤＰＨ保温后，人脑ＡＬＲ纯品的活性与对照酶组相似，且不被糖酵解途径的一些磷酸化中间产物抑制．苯基硼酸琼脂糖柱层析洗脱谱峰和氢硼化钠还原反应提示，当同葡萄糖保温时，人脑ＡＬＲ（特别是其均一态）可能未被进一步糖基化．在糖尿病并发症和按结构完成药物设计的研究工作中。

Aldose reductase(EC1.1.1.21;ALR)from human brain was purified by combination of DEAE cellulose chromatography,chromatofocusing,NADP affinity chromatography and Sephadex G 100 gel filtration.The isoelectric pH for this enzyme was determined to be 5.85.The high purity of the enzyme was confirmed by disc PAGE and Western blotting.The purified human brian ALR after incubation with glucose,glucose 6 phosphate and NADPH was comparable to the control enzyme in activity,and was not inhibited by several phosphorylated intermediates of glycolytic pathway.Phenylboronate agarose column profile and sodium borohydride reduction imply that human brain ALR,especially at the homogenous state,is possibly not further glycosylated during incubation with glucose. Application of purified ALR is important for studying the pathogenesis of diabetic complications and structure based drug design.