Wise L S, Rubin C S
Anal Biochem. 1984 Jul;140(1):256-63. doi: 10.1016/0003-2697(84)90162-3.
A very rapid and highly effective procedure has been devised for the isolation of homogeneous malic enzyme from rat liver cytosol. A combination of precipitation with 10 to 20% polyethylene glycol, ion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Procion Red HE-3B Agarose was used to prepare 3 to 4 mg of homogeneous malic enzyme from the livers of two rats in 18 h. In addition to introducing the advantages of simplicity, speed, and high yield (31%) the new method eliminates potentially denaturing steps (heat treatment, ethanol fractionation) and prolonged dialysis procedures used in other purification schemes. Malic enzyme purified by this new method was use to immunize rabbits. The resulting antibodies bound purified rat liver and mouse liver malic enzymes with very similar affinities and also avidly complexed cytosolic malic enzyme from two murine cell lines, 3T3-L1 preadipocytes and 3T3-C2 fibroblasts. When purified malic enzyme was incubated with lactoperoxidase, glucose oxidase and Na 125I 1.8 atoms of 125I were incorporated per molecule of enzyme with full retention of catalytic activity, subunit size, and immunoreactivity. The antiserum, the purified enzyme, and enzymatically iodinated 125I-malic enzyme were used to construct a sensitive, competitive binding radioimmunoassay for the measurement of malic enzyme mass in the range of 1 to 100 ng.
已设计出一种非常快速且高效的程序，用于从大鼠肝脏胞液中分离纯化苹果酸酶。该程序结合了用10%至20%聚乙二醇沉淀、在DEAE - 纤维素上进行离子交换色谱以及在Procion Red HE - 3B琼脂糖上进行亲和色谱，在18小时内从两只大鼠的肝脏中制备出3至4毫克纯化的苹果酸酶。除了具有操作简单、速度快和产量高（31%）的优点外，新方法还避免了其他纯化方案中可能导致变性的步骤（热处理、乙醇分级分离）以及冗长的透析程序。用这种新方法纯化的苹果酸酶用于免疫兔子。所得抗体以非常相似的亲和力结合纯化的大鼠肝脏和小鼠肝脏苹果酸酶，并且还能与两种鼠细胞系（3T3 - L1前脂肪细胞和3T3 - C2成纤维细胞）的胞液苹果酸酶紧密结合形成复合物。当将纯化的苹果酸酶与乳过氧化物酶、葡萄糖氧化酶和Na 125I一起孵育时，每分子酶掺入1.8个125I原子，同时酶的催化活性、亚基大小和免疫反应性完全保留。该抗血清、纯化的酶以及经酶促碘化的125I - 苹果酸酶被用于构建一种灵敏的竞争性结合放射免疫测定法，用于测量1至100纳克范围内的苹果酸酶质量。
