Conrad S M, Doughty C C
Biochim Biophys Acta. 1982 Nov 19;708(3):348-57. doi: 10.1016/0167-4838(82)90448-4.
A purification scheme for aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) developed using bovine lens tissue including an affinity chromatographic step is presented which is particularly suited for small quantities of lenses. Using the affinity chromatographic method as a key step also makes it possible to obtain preparations of rat lens aldose reductase which are homogeneous. The behavior of crude preparations of aldose reductase from human lens on both ion-exchange and affinity chromatography was similar to the chromatographic behavior of the enzyme from rat and bovine lens. Comparative studies of aldose reductase obtained from the lenses of the three species demonstrate the similarity of the enzymes. These comparisons were based on molecular weights, isoelectric points, chromatographic behavior and kinetic data. Homotropic cooperativity for both NADPH and glyceraldehyde, as evidenced by a downward curvature in the Lineweaver-Burk double-reciprocal plots, had been demonstrated for aldose reductase obtained from bovine lens (Sheaff, C.M. and Doughty, C.C. (1976) J. Biol. Chem. 251, 2696-2702). Similarly, cooperativity was observed with the enzyme from both rat and human lenses and the apparent Km values at both high and low concentrations of substrate are comparable for the lens aldose reductases from all three species for both substrates.
本文介绍了一种利用牛晶状体组织开发的醛糖还原酶（醛糖醇：NADP+ 1-氧化还原酶，EC 1.1.1.21）纯化方案，该方案包括一个亲和色谱步骤，特别适用于少量晶状体。将亲和色谱法作为关键步骤，也能够获得纯的大鼠晶状体醛糖还原酶制剂。人晶状体醛糖还原酶粗制品在离子交换色谱和亲和色谱上的行为与大鼠和牛晶状体中该酶的色谱行为相似。对从这三个物种的晶状体中获得的醛糖还原酶的比较研究表明了这些酶的相似性。这些比较基于分子量、等电点、色谱行为和动力学数据。从牛晶状体中获得的醛糖还原酶已证明对NADPH和甘油醛均有同促协同作用，这在Lineweaver-Burk双倒数图中表现为向下弯曲（Sheaff, C.M.和Doughty, C.C.（1976年）《生物化学杂志》251, 2696 - 2702）。同样，在大鼠和人晶状体的酶中也观察到了协同作用，并且对于所有三个物种的晶状体醛糖还原酶，两种底物在高浓度和低浓度下的表观Km值相当。
