Dons R F, Doughty C C
Biochim Biophys Acta. 1976 Nov 8;452(1):1-12. doi: 10.1016/0005-2744(76)90053-x.
Aldose reductase activity (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) from calf brain was separated into two protein fractions by DEAE chromatography. Further purifcation by molecular sieve chromotography and electrofocusing yielding two distinctive enzymes, which were designated AR I and AR II. AR I was purified 646-fold and found to have an isoelectric point of 6.18. AR I was most active as a monomer with a molecular weight of 29 000 and appeared to be in equilibrium with a less active dimer. AR II was purified 425-fold and found to have an isoelectric point of 4.88. The molecular weight of this enzyme was 30 000. Although both enzymes had specificity for aldoses as substrates, AR I had two to three times larger turnover numbers with aromatic aldehydes and hexonates than did AR II. AR I was activated by sulfhydryl compounds and exhibited biphasic double reciprocal plots. AR I was more sensitive to inhibition by high substrate and phenobarbital concentrations than was AR II. AR I and AR II did not have antigenic similarity as tested by Ouchterlony immunodiffusion and counter immunoelectrophoresis. An immunochemical cross-reaction was observed between AR II and lens aldose reductase.
通过DEAE柱色谱法，从小牛脑中分离出醛糖还原酶活性（醛糖醇：NADP + 1-氧化还原酶，EC 1.1.1.21），得到两个蛋白质组分。通过分子筛色谱法和等电聚焦进一步纯化，得到两种不同的酶，分别命名为AR I和AR II。AR I纯化了646倍，其等电点为6.18。AR I作为单体时活性最高，分子量为29000，并且似乎与活性较低的二聚体处于平衡状态。AR II纯化了425倍，其等电点为4.88，该酶的分子量为30000。虽然两种酶都对醛糖具有底物特异性，但AR I对芳香醛和己糖醛酸的转换数比AR II大两到三倍。AR I被巯基化合物激活，并呈现双相双倒数图。与AR II相比，AR I对高底物浓度和苯巴比妥浓度的抑制更敏感。通过双向免疫扩散和对流免疫电泳检测，AR I和AR II没有抗原相似性。在AR II和晶状体醛糖还原酶之间观察到免疫化学交叉反应。
