Wallin R, Gebhardt O, Prydz H
Biochem J. 1978 Jan 1;169(1):95-101. doi: 10.1042/bj1690095.
A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
建立了一种简单的三步法，通过亲和色谱从大鼠肝脏中纯化NAD(P)H脱氢酶（醌）（“DT-黄递酶”，EC 1.6.99.2），回收率超过50%。最终的酶制剂纯化了约750倍，且在电泳上呈均一性。凝胶过滤显示该酶的分子量约为55000，每55000分子量含有一分子FAD。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测得分子量约为27000。发现两种N端氨基酸，天冬酰胺/天冬氨酸和谷氨酰胺/谷氨酸，产量大致相等，这表明该酶中存在两条不同的多肽链。通过这种亲和色谱方法可从微粒体羧化系统中选择性去除NAD(P)H脱氢酶。该系统用去污剂溶解，依赖维生素K（2-甲基-3-植基-1,4-萘醌或带有其他侧链的类似物），去除该酶后失去活性。通过添加纯化的细胞质NAD(P)H脱氢酶或使用维生素K1的氢醌形式（2-甲基-3-植基-1,4-萘氢醌），可使该系统的活性完全恢复。
