17 beta-Hydroxysteroid dehydrogenase of the sheep ovary : purification, properties and substrate binding site.
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.
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology*; D012756:Sheep; D012967:Sodium Dodecyl Sulfate; D013329:Structure-Activity Relationship; D013696:Temperature
D012967:Sodium Dodecyl Sulfate; D004958:Estradiol; D006913:Hydroxysteroid Dehydrogenases