Physical and kinetic properties of homogenous bovine lens aldose reductase.
Physical and kinetic properties of homogenous bovine lens aldose reductase.
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均质牛晶状体醛糖还原酶的物理和动力学特性。
DOI：
10.1016/s0021-9258(17)33543-3
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
C. C. Doughty
中科院分区：
文献类型：
--
作者：
C. Sheaff;C. C. Doughty
小牛透镜的醛糖还原酶纯化了15，000倍。通过分子筛色谱、分析超离心、十二烷基硫酸钠凝胶电泳、Ouchterlony免疫扩散和聚丙烯酰胺凝胶电泳在三种pH值下证明了最终制剂的均一性。分子筛层析和β-巯基乙醇十二烷基硫酸钠凝胶电泳的分子量均为37，000，表明该酶为单体性质。这与通过使用Svedberg方程中的计算物理常数估计的40，400的分子量密切对应。根据超浓缩和蔗糖密度梯度数据确定的S_（20），w为3.6 - 3.7。通过两种不同的方法发现斯托克斯半径为2.5 +/- 0.2 nm和2.75 +/- 0.15 nm。扩散常数D20，w为（7.8 +/-10（-7）+/-0.45 X 10（-7）cm 2/s）。该分子几乎是球形的，如摩擦比f/fo = 1.14所示。从圆二色性数据估计α-螺旋含量为5%，并且在添加底物、产物和一些酶抑制剂的存在下没有变化。同向性协同效应的观察所示的凹向下曲率的倒数图。
Aldose reductase from calf lens was purified 15,000-fold. The homogeneity of the final preparation was demonstrated by molecular sieve chromatography, analytical ultracentrifugation, sodium dodecyl sulfate gel electrophoresis, Ouchterlony immunodiffusion, and polyacrylamide gel electrophoresis at three pH values. The monomeric nature of the enzyme is suggested by the molecular weight of 37,000 from both molecular sieve chromatography and sodium dodecyl sulfate-gel electrophoresis with beta-mercaptoethanol. This closely corresponds with a molecular weight of 40,400 estimated by using calculate physical constants in the Svedberg equation. The S20,w was 3.6 to 3.7 as determined from ultracentrifuge and sucrose density gradient data. The Stokes radius was found to be 2.5 +/- 0.2 nm and 2.75 +/- 0.15 nm by two different methods. The diffusion constant D20,w is (7.8 +/- 10(-7) +/- 0.45 X 10(-7) cm2/s). The molecule is nearly spherical as indicated by a frictional ratio f/fo = 1.14. The alpha-helical content was estimated from circular dichroism data to be 5% and did not change in the presence of added substrates, products, and some enzyme inhibitors. Homotropic cooperative effects were observed as shown by the concave downward curvature of the reciprocal plots.