Michel F, Nicolas J C, de Paulet A C
Biochimie. 1975;57(10):1131-40. doi: 10.1016/s0300-9084(76)80574-3.
Sheep ovarian 17 beta HSDH has been purified about 1000 fold to a specific activity of 0.5 IU/mg protein, using DEAE cellulose chromatography, affinity chromatography on estrone-amino caproate-Sepharose and a second DEAE cellulose chromatography. The molecular weight is 70,000 ; the pH optimum for activity is 9.2 and the energy of activation is 16.5 Kcal/mole. The kinetics of the oxidation of estradiol and many analogues have been studied at various concentrations and in the presence of different amounts of coenzyme. The data are in agreement with a compulsory order mechanism with the binding of NAD+ as the first substrate. Sheep ovarian 17 beta HSDH accepts subtituents in position C3, C11, C13 ; the substrate binding site is open in this region. On the contrary, the binding requirements are strict for the region of C10 since the presence of a C19 methyl group impairs binding and (or) oxidation of the steroid. Sheep ovarian and human placental 17 beta HSDH have close analogies : molecular weight, pH optimum, substrate binding site requirements. Their reaction mechanisms are different : random for the placental 17 beta HSDH, compulsory order for the ovarian 17 beta HSDH : this can be explained by the effect of the coenzyme upon the binding of the substrate : without effect on placental enzyme, the coenzyme fixation enhances the affinity of the ovarian 17 beta HSDH for any substrate.
利用二乙氨基乙基纤维素色谱法、雌酮 - 氨基己酸 - 琼脂糖亲和色谱法以及第二次二乙氨基乙基纤维素色谱法，绵羊卵巢17β - 羟类固醇脱氢酶（17β - HSDH）已被纯化约1000倍，比活性达到0.5 IU/mg蛋白质。其分子量为70,000；活性的最适pH为9.2，活化能为16.5千卡/摩尔。已在不同浓度以及存在不同量辅酶的情况下研究了雌二醇及其许多类似物的氧化动力学。数据符合以NAD⁺作为第一个底物结合的强制顺序机制。绵羊卵巢17β - HSDH在C3、C11、C13位接受取代基；该区域的底物结合位点是开放的。相反，对于C10区域的结合要求则很严格，因为C19甲基的存在会损害类固醇的结合和（或）氧化。绵羊卵巢17β - HSDH和人胎盘17β - HSDH有密切的相似之处：分子量、最适pH、底物结合位点要求。它们的反应机制不同：胎盘17β - HSDH是随机机制，卵巢17β - HSDH是强制顺序机制：这可以通过辅酶对底物结合的影响来解释：辅酶对胎盘酶没有影响，而辅酶的结合增强了卵巢17β - HSDH对任何底物的亲和力。
