Mizunaga T, Katakura Y, Miura T, Maruyama Y
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo.
J Biochem. 1990 Nov;108(5):846-51. doi: 10.1093/oxfordjournals.jbchem.a123291.
Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from Saccharomyces cerevisiae. The enzyme was purified 1,850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent Mr of 140,000 on gel filtration chromatography, and its NH2-terminal was blocked. The Mr of its subunits were estimated to be 70,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The Mr of the subunits changed to 60,000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The Km values of yeast and bovine PDIs for scrambled RNase were 1 x 10(-5) and 2 x 10(-5) M, and their Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in Km or Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.
从酿酒酵母中纯化出了能使无活性的混乱核糖核酸酶重新激活的蛋白质二硫键异构酶（PDI）。通过五个纯化步骤将该酶纯化了1850倍，达到表观均一性：30 - 70%硫酸铵分级沉淀、DEAE Toyopearl - 650S和丁基Toyopearl - 650S层析，以及含或不含半胱氨酸的差异苯基 - 5PW高效液相色谱。在凝胶过滤色谱上，天然酶的表观分子量为140,000，其氨基末端被封闭。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计其亚基的分子量为70,000，表明该酶可能由两个相同的亚基组成。经内切葡糖胺酶H处理后，亚基的分子量变为60,000，表明该酶被转运到内质网中。该酶的最适pH为8.5，pI为4.02。将其酶学性质与纯化的牛肝PDI的酶学性质进行了比较。酵母和牛PDI对混乱核糖核酸酶的Km值分别为1×10⁻⁵和2×10⁻⁵ M，它们的Vmax值分别为6和7单位/毫克蛋白质。这两种酶在硫醇化合物的Km或Vmax值方面没有显著差异。杆菌肽以相同方式抑制这两种PDI。这些结果表明这种酵母PDI与哺乳动物PDI相对应。
