Rapid purification and radioimmunoassay of cytosolic malic enzyme.
Rapid purification and radioimmunoassay of cytosolic malic enzyme.
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胞质苹果酸酶的快速纯化和放射免疫分析。
DOI：
10.1016/0003-2697(84)90162-3
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发表时间：
1984
影响因子：
2.9
通讯作者：
Rubin,CS
中科院分区：
文献类型：
--
作者：
Wise,LS;Rubin,CS
本文提出了一种从大鼠肝胞液中分离均质苹果酸酶的快速、高效方法。用10 - 20%聚乙二醇沉淀、在DEAE-纤维素上的离子交换层析和在Procion Red HE-3B琼脂糖上的亲和层析的组合用于在18小时内从两只大鼠的肝脏制备3 - 4 mg均质苹果酸酶。除了引入简单、快速和高产率（31%）的优点外，新方法还消除了其他纯化方案中使用的潜在变性步骤（热处理、乙醇分馏）和延长的透析程序。用此方法纯化的苹果酸酶免疫家兔。所得到的抗体结合纯化的大鼠肝脏和小鼠肝脏苹果酸酶具有非常相似的亲和力，也积极复杂的细胞溶质苹果酸酶从两个鼠细胞系，3 T3-L1前脂肪细胞和3 T3-C2成纤维细胞。当纯化的苹果酸酶孵育乳过氧化物酶，葡萄糖氧化酶和Na 125 I 1.8原子的125 I被纳入每分子酶的催化活性，亚基大小，和免疫反应性的充分保留。抗血清，纯化的酶，和酶碘化125 I-苹果酸酶被用来构建一个敏感的，竞争性结合放射免疫测定苹果酸酶质量的测量范围为1至100纳克。
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 used 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 Na125I 1.8 atoms of125I were incorporated per molecule of enzyme with full retention of catalytic activity, subunit size, and immunoreactivity. The antiserum, the purified enzyme, and enzymatically iodinated125I-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.