Immunological measurement of human 17 beta-hydroxysteroid dehydrogenase.
Human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) was purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on Red-Agarose and DEAE-Sepharose columns. Electrophoresis on polyacrylamide gels under denaturing conditions and using silver staining showed a single protein with an apparent molecular weight of 37,800. Antibodies to the purified protein were raised in rabbits and were found by immunoblotting to be specific to 17-HSD. A sensitive radioimmunoassay was established using 125I-labeled 17-HSD as a tracer, an appropriate dilution of the antibody, and a kaolin-coupled double antibody for separating the antibody-bound and free fractions. The detection limit of the assay was approximately 150 pg/tube (1.5 micrograms/l). The cytosol fraction (105,000 g) of term placental tissue contained approximately 0.7 mg of 17-HSD per gram of protein, and the concentrations of 17-HSD measured by immunoassay and enzymatic activity proved to be strictly parallel in different partly purified placental preparations. The supernatants from centrifugations of human endometrial homogenates at 800 g and 105,000 g (after detergent treatment) displayed cross-reactivity with the antibody. The mean concentration of the cross-reacting substance in the radioimmunoassay was 14.1 micrograms/g protein (range 2-62.3) in specimens taken on different days in the cycle. These concentrations showed a significant correlation with the 17-HSD activities measured in the endometrial specimens (r = 0.722, P less than 0.001, n = 21). Mean concentrations of substance were 8.3 micrograms/g protein in endometrial specimens taken during the follicular phase (days 4-12, n = 8) and 22.9 micrograms/g protein during the luteal phase (days 16-22, n = 6) were obtained using the radioimmunoassay. There was excellent parallelism between the competition curves for [125I]iodo-17-HSD with purified 17-HSD standards and placental and endometrial homogenate dilutions. These data strongly suggest that the substance measured in the endometrial specimens was 17-HSD.
Mäentausta
O
O
Department of Clinical Chemistry, University of Oulu, Finland.
Peltoketo
H
H
Isomaa
V
V
Jouppila
P
P
Vihko
R
R
D015067:17-Hydroxysteroid Dehydrogenases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D000937:Antigen-Antibody Reactions; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007553:Isotope Labeling; D010920:Placenta / Q000201:enzymology* / Q000276:immunology; D011863:Radioimmunoassay; D013347:Subcellular Fractions / Q000201:enzymology; D014594:Uterine Neoplasms / Q000201:enzymology
D000906:Antibodies; D015067:17-Hydroxysteroid Dehydrogenases