Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells.
Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.
Adamski
J
J
Max-Planck-Institute für experimentelle Endokrinologie, Hannover, Germany.
Husen
B
B
Marks
F
F
Jungblut
P W
PW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002845:Chromatography; D003594:Cytoplasmic Granules / Q000201:enzymology; D004717:Endometrium / Q000201:enzymology*; D004960:Estradiol Dehydrogenases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D007425:Intracellular Membranes / Q000201:enzymology; D008970:Molecular Weight; D013379:Substrate Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D004960:Estradiol Dehydrogenases