Hino Y, Minakami S
J Biol Chem. 1982 Mar 10;257(5):2563-8.
Hexose-6-phosphate dehydrogenase was purified from rat liver microsomal fraction more than 500-fold with a 45% recovery using DEAE-cellulose and 2',5'-ADP-Sepharose 4B columns. The purified enzyme appeared to be immunologically and electrophoretically homogeneous and had broad substrate and cofactor specificities. The enzyme activity was not inhibited by p-chloromercuribenzoate. The purified enzyme was a glycoprotein in nature, having a Stokes radius of about 55 A, a sedimentation coefficient of about 8.2 s, and an isoelectric point of about 6.4. Minimum molecular weight of the enzyme was about 108,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the product cross-linked with glutaraldehyde or dimethyl suberimidate had Mr approximately equal to 220,000, suggesting that the active enzyme existed as a dimer of identical subunits. Antiserum raised against the purified enzyme inhibited the activity of the solubilized enzyme but did not inhibit the cytosol glucose-6-phosphate dehydrogenase activity. The antigenic sites of the enzyme were latent in intact microsomes. Comparison was also made between the enzymes isolated from untreated and phenobarbital-pretreated animals.
利用二乙氨基乙基纤维素（DEAE-纤维素）和2',5'-二磷酸腺苷琼脂糖4B柱，从大鼠肝脏微粒体组分中纯化出己糖-6-磷酸脱氢酶，纯化倍数超过500倍，回收率为45%。纯化后的酶在免疫学和电泳方面表现为均一性，具有广泛的底物和辅因子特异性。该酶活性不受对氯汞苯甲酸的抑制。纯化后的酶本质上是一种糖蛋白，斯托克斯半径约为55埃，沉降系数约为8.2 s，等电点约为6.4。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，该酶的最小分子量约为108,000，而与戊二醛或辛二酸二甲酯交联后的产物分子量约为220,000，这表明活性酶以相同亚基的二聚体形式存在。针对纯化酶产生的抗血清抑制了可溶酶的活性，但不抑制胞质葡萄糖-6-磷酸脱氢酶的活性。该酶的抗原位点在完整的微粒体中是潜在的。还对从未经处理和经苯巴比妥预处理的动物中分离出的酶进行了比较。
