H. Kappus, H. Bolt, H. Remmer
Aug 1, 1973
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0
Influential Citations
78
Citations
Quality indicators
Journal
Steroids
Abstract
Abstract During incubation of 6,7-3H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak. The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding. Further evidence resulted from experiments in vivo , dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after 3H-estradiol.