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Androgen and progesterone binding components in cytosol prepared from cultures enriched in Sertoli cells from immature rat testes
Journal article   Open access   Peer reviewed

Androgen and progesterone binding components in cytosol prepared from cultures enriched in Sertoli cells from immature rat testes

W N Schmidt and B J Danzo
Biology of reproduction, Vol.23(3), pp.495-506
10/1980
DOI: 10.1095/biolreprod23.3.495
PMID: 6160880
url
https://doi.org/10.1095/biolreprod23.3.495View
Published (Version of record) Open Access

Abstract

Sephadex G-25 chromatography has been used to examine cytosol prepared from 5–6-day-old cultures of immature rat Sertoli cells for the presence of macromolecular androgen and progesterone binding components. High affinity, slowly dissociable, androgen binding activity was detected that exhibited first-order half-times of dissociation (t½) of 3.2 ± 1.0 h for [3H]-testosterone and 3.5 ± 1.3 h for [3H]-5α-dihydrotestosterone. These dissociation rates clearly distinguish this androgen binding component from Androgen Binding Protein (ABP), which has a t½ of ∿5 min. Specific binding of [3H]-testosterone to the non-ABP binding moiety was completely inhibited by a 100-fold molar excess of unlabeled testosterone, 5α-DHT, Δ6-testosterone, and progesterone. Specific binding of [3H]-progesterone was also observed. Approximately 20%–40% of the specific binding persisted 30 min after the addition of 100-fold molar excess of unlabeled progesterone to samples that had been equilibrated with [3H]-progesterone. Analysis of the dissociation of [3H]-progesterone bound to macromolecules with time revealed the presence of at least two binding components. One component showed a rapid rate of dissociation (t½ ∿ 13 min), and the other component showed a slow rate of dissociation (t½>5 h). Specific binding of [3H]-progesterone to the slowly and rapidly dissociating components showed similar but not identical sequences of binding specificity (slow component: P>5α-DHT>T = Δ6-T>R5020>MPA = E = F; rapid component: P>5α-DHT>R5020>T = 6Δ-T>MPA = E = F). Studies on the metabolism of [3H]-5α-DHT, [3H]-testosterone and [3H]-progesterone by Sertoli cell cytosol (3 h, 0°C-2°C) revealed that: 1) ∿50% of the [3H]-5α-DHT was converted to [3H]-5α-androstan-3α, 17β-diol, and 2) little (<5%) metabolism of [3H]-testosterone or (3H]-progesterone occurred. When the radioactivity specifically bound to macromolecular components in cytosol was examined, we determined that: 1) in incubations with [3H]-5α-DHT, 90% of the bound label was [3H]-5α-DHT; 2) in incubations with [3H]-testosterone, greater than 94% of the bound label was [3H]-testosterone; and 3) in incubations with [3H]-progesterone, greater than 98% of the bound label was [3H]-progesterone.
 Binding, Competitive Progesterone-Binding Globulin - analysis Sertoli Cells - metabolism Radioisotope Dilution Technique Cells, Cultured Rats Male Hot Temperature Testosterone - metabolism Animals Alpha-Globulins - analysis Progesterone - metabolism Cytosol - metabolism Androgens - metabolism Carrier Proteins - analysis Chromatography, Gel Androgen-Binding Protein - analysis

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