Human aldehyde dehydrogenase: improved purification procedure and comparison of homogeneous isoenzymes E1 and E2.
Human aldehyde dehydrogenase: improved purification procedure and comparison of homogeneous isoenzymes E1 and E2.
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人醛脱氢酶：改进的纯化程序以及同工酶 E1 和 E2 的比较。
DOI：
10.1111/j.1530-0277.1982.tb05001.x
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Pietruszko,R
中科院分区：
文献类型：
--
作者：
Hempel,JD;Reed,DM;Pietruszko,R
本文介绍了一种改进的人乙醛脱氢酶（EC 1.2.1.3）同工酶E1和E2的纯化方法。该方法仅采用三个色谱步骤，以60%的总产率生产均一的E1和E2同工酶。同工酶已测试的同质性电泳的本地和变性物种，比活性测定后再层析，以及映射的胰蛋白酶和氯化萘片段。还对每种同工酶进行了总SH基团分析。结果表明，这两种同工酶是均一的。E1和E2同工酶之间的相似性，注意到在约40%的胰蛋白酶片段，总SH含量，和两个CNBr片段的流动性的流动性。结果还表明，在CNBr图谱中，E1和E2的不同分子量的片段和约60%的胰蛋白酶片段迁移到不同的位置，同工酶之间存在相当大的结构差异。只有一个含SH的胰蛋白酶片段迁移到两种同工酶的相同位置。E1和E2均由亚基组成，在十二烷基硫酸钠（SDS）和尿素电泳中均以单一条带迁移。E1和E2亚基在SDS凝胶中的迁移率相似，但在尿素中的迁移率不同，表明E1亚基与E2亚基不同，同工酶不共享亚基。因此，同工酶之间的结构相似性必然是由组成E1和E2分子的不同多肽链区域内的序列相似性引起的。这些结果提供了一个制备同质同工酶的简化程序，它们还表明，E1和E2是不同基因的产物，这些基因可能是通过基因复制和细胞的区室化从一个共同的遗传祖先中分化出来的。
An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1and E2is presented. This procedure employs only three chromatographic steps to produce homogeneous E1and E2isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1and E2isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1and E2of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH‐containing tryptic fragments migrates to the same location in both isoenzymes. E1and E2each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1and E2subunits in SDS gels is similar, it is different in urea, showing that subunits of E1are distinct from those of E2and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1and E2molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1and E2are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell.