Estradiol-17 beta dehydrogenase from chicken liver.
NADP+-linked estradiol-17 beta dehydrogenase has been purified 300- to 400-fold from cell-free extracts of chicken liver in a 20 to 30% yield by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The enzyme is stable for at least 3 months when stored at -20 degrees C in buffer containing glycerol (50%, v/v). Two forms, with molecular weights of 43,000 and 97,000 are present; these show one major band (Rm = 0.27) and one minor band (Rm = 0.25) on polyacrylamide disc gel electrophoresis. (Rm is defined as the ratio of the distance migrated by the protein band to that of the tracking dye.) The species of lower molecular weight is the more active, with apparent Km values for estradiol-17 beta of 25 and 17.3 microM in the presence and absence, respectively, of bovine serum albumin in the assay medium. The apparent Km for NADP+ is 7.7 microM, and the optimum pH for dehydrogenation is 9.9. The lower molecular weight form has a lambda max at 280 nm, a shoulder at 290 nm, and an A 1% 1 cm of 12.1 at 280 nm. The fluorescence spectrum corresponds to that of a tryptophan-containing protein with lambda max at 288 nm. Isoelectric focusing in gel at pH 5 to 8 shows three major bands of pI 6.9, 6.8, and 6.0. Cross-linking with dimethyl suberimidate followed by electrophoresis reveals five bands. The enzyme is affected by thio reagents and possesses no associated estradiol-sensitive transhydrogenase activity.
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004960:Estradiol Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity
D002729:Chloromercuribenzoates; D046911:Macromolecular Substances; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D004229:Dithiothreitol