A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

Mainwaring, W.I.P.; Peterken, Brenda M.

doi:10.1042/bj1250285

Cited by 178 publications

(61 citation statements)

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“…1). This confirms the results of other investigators (15,16 (7,20). The Both these components (A and B) exhibited high affinity and specificity for progesterone, and both associated with isolated chick oviduct nuclei.…”

Section: Resultssupporting

confidence: 81%

“…The active fraction was freed from unbound hormone by gel -filtration on a Sephadex G-25 column (1 X 24 cm). Sucrose density gradient analysis of the purified [3H]DHT-receptor complex showed that essentially all the radioactivity was associated with the receptor protein peak sedimenting at approximately 8 S. This is in agreement with the reports from other laboratories (15,16). In our system, we were able to detect only a small amount of 3.5S receptor complex, probably because of the (NH4)2SO4 fractionation of the initial extract (16 Samples (1 ml of reconstituted chromatin with [3H]DHTreceptor complex) were analyzed by density gradient centrifugation.…”

supporting

confidence: 82%

“…The reconstitution was accomplished by mixing the interacting components in 2.5 M NaCl, 5.0 M urea, 50 mM Tris-HCl buffer, pH 8.0, at a ratio of DNA:UP:HP:NP = 1:1: 1:0.2 and decreasing the NaCl concentration by a slow dialysis against 5.0 M urea in 50 mM Tris.HCl buffer (13). Finally, the urea was removed by dialysis against either 0.1 X SSC (SSC = standard saline citrate: 150 mM NaCl, 15 mM sodium citrate), or 10 mM Tris-HCl buffer, pH 7.6. The reconstituted chromatins were used in hormone acceptor experiments.…”

mentioning

confidence: 99%

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Acceptor proteins in rat androgenic tissue chromatin.

Klyzsejko-Stefanowicz¹,

Chiu²,

Tsai³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Fractionation of chromatin into urea-soluble chromosomal nonhistone proteins (UP), histones (HP), and DNA-associated nonhistone proteins (NP) revealed that the NP fraction from testicular and prostatic chromatin contains organ-specific acceptors for complexes of 5a-dihydrotestosterone (17j3-hydroxy-5a-androstan-3-one) and its receptor. This acceptor capacity of androgenic tissue chromatin could be transferred to chromatins from non-target tissues with the NP fraction of DNA-associated proteins. Phosphorylation of chromatin enhanced its hormone-receptor binding capacity. According to current views, early steps in the action of steroid hormones on their target tissues involve the association of the steroid hormone with cytoplasmic receptor, transport of this complex from cytoplasm into the nucleus, and, finally, its association with specific acceptor sites on chromatin (1-4). This association alters the transcriptional controls and allows the synthesis of new mRNA species. The new mRNAs are eventually translated into new proteins that are often specific for the hormone-stimulated tissue (1,3,5,6). The target cell nuclear acceptor is selective in that the nuclei of responsive cells accept more hormone-cytosol protein complex than the nuclei of tissues refractory to the action of steroid hormones (7,8). Recent years have seen the accumulation of considerable information concerning the steroid hormone-cytoplasmic receptor interactions in different types of tissue. After the hormone interacts with cytoplasmic receptor, the hormone-protein complex moves to the nucleus, where it is incorporated into chromatin. Liao and Fang (1) coined the term "acceptor protein" for the nuclear acceptor molecule. The acceptor protein fraction was isolated from prostate (9) and found to be heat labile and tissue specific, and could be transferred from the chromatin of one tissue to another with the fraction of chromosomal nonhistone proteins (3, 7). We report here the results of our studies on chromatin acceptor(s) in rat prostate and testis. MATERIALS AND METHODSProstatp, testis, liver, and pancreas from male Sprague-Dawley rats (125-150 g) were homogenized in ice-cold 0.25 M sucrose, 5 mM MgCl2, and 2 mM 2-mercaptoethanol in 20 mM Tris-HCI buffer, pH 6.8. The homogenate was centrifuged at 800 X g for 10 min. The crude nuclear pellet was purified by rehomoAbbreviations: SSC, standard saline citrate (150 mM NaCl, 15 mM sodium citrate); UP, chromosomal nonhistone proteins soluble in urea at low ionic strength; HP, histones; NP, DNA-binding chromosomal nonhistone protein fraction; DHT, 5a-dihydrotestosterone (17f,-hydroxy-5a-androstah-3-one). After incubation with labeled hormone, the cytosol-hormone complex was fractionated with (NH4)2SO4 (0-33% saturation). The active fraction was freed from unbound hormone by gel -filtration on a Sephadex G-25 column (1 X 24 cm). Sucrose density gradient analysis of the purified [3H]DHT-receptor complex showed that essentially all the radioactivity was associated with the receptor protein peak ...

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Section: Resultssupporting

confidence: 81%

supporting

confidence: 82%

mentioning

confidence: 99%

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Acceptor proteins in rat androgenic tissue chromatin.

Klyzsejko-Stefanowicz¹,

Chiu²,

Tsai³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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“…unloading). However, there is no evidence to support this hypothesis; rather, it has been demonstrated (Mainwaring & Peterken, 1971;Rennie & Bruchovsky, 1973; that administration of androgen results in a net increase in the concentration of androgen receptors in the nucleus that is stoicheiometrically equivalent to a net decrease of androgen receptors in the cytoplasm. Although the second hypothesis cannot be completely discounted, two points argue against its acceptance: first, preparation of nuclei in the presence of a proteinase inhibitor alters the intranuclear distribution of specific dihydrotestosterone-binding sites but not their recovery (20600 sites per nucleus; Barrack & Coffey, 1980); secondly, even with very high reported estimates for nuclear androgen receptors (26000 sites per nucleus) the ratio of total nuclear dihydrotestosterone to receptor is too high (5: 1) to be due to recovery alone (Bruchovsky et al, 1980).…”

Section: Effects Of Dose On the Intracellular Concentration Of Dihydrmentioning

confidence: 90%

Radioimmunoassay measurements of nuclear dihydrotestosterone in rat prostate. Relationship to androgen receptors and androgen-regulated responses

Larminat¹,

Rennie²,

Bruchovsky³

1981

Biochemical Journal

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The concentration of dihydrotestosterone was measured by radioimmunoassay in nuclear and cytoplasmic extracts from rat ventral prostates. In the regenerating prostates of castrated rats treated with dihydrotestosterone for 4 days, the nuclear concentration of this steroid increased from approx. 70nM to 800nM as a linear function of the injected dose, whereas the cytoplasmic concentration remained relatively constant (70-130nM). Isotope-exchange measurements of nuclear androgen receptors by using [3H]methyltrienolone indicated that, although the concentration of nuclear dihydrotestosterone was several-fold higher than the concentration of androgen receptors, they were logarithmically related. The recruitment of prostatic cells into the growth fraction and the stimulation of 5 a-reductase activity were more directly correlated to the nuclear concentration of androgen receptors than to the total nuclear concentration of dihydrotestosterone. Maximal restoration of a specific isoenzyme of acid phosphatase was achieved when approx. 2000 androgen receptors were present in the prostatic nuclei; higher concentrations of nuclear androgen receptors were associated with decreased amounts of this enzyme. Hence the results imply, first, that the total amount of dihydrotestosterone accumulated by nuclei is not a direct consequence of carrier-mediated transport by androgen receptors, and, secondly, that, whereas acid phosphatase may be differentially controlled by androgens in the regenerating prostate, increases in the amount of cell proliferation and 5a-reductase activity directly parallel increases in the nuclear concentration of androgen receptors.In the prostate, androgenic regulation of physiological functions and cellular growth is attributed largely to the formation, and subsequent binding to chromatin, of androgen-receptor complexes (King & Mainwaring, 1974 (Coffey, 1974;Rennie et al., 1975;Van Doorn et al., 1976). Although it has been postulated that androgen receptors are involved in the uptake and retention of dihydrotestosterone by target-cell * To whom correspondence should be addressed.Vol. 200 nuclei (Rennie & Bruchovsky, 1973;Bruchovsky et al., 1975a), a direct stoicheiometric relationship has not been demonstrated. When castrated rats are injected with radioactive androgen in the ventral prostate the concentration of radioisotope measured in nuclei exceeds that measured in the cytoplasm (Bruchovsky et al., 1975b). However, the concentration of androgen-receptor complexes recovered from the nuclei is insufficient to account for the nuclear influx of androgens if a mole-to-mole relationship is assumed. Furthermore, radioimmunoassay measurements of dihydrotestosterone in ventral prostates from non-castrated rats have indicated that only about 20% of the endogenous nuclear androgen is bound to androgen receptors (Bruchovsky et al., 1980). Whether the large nuclear concentration of non-receptor-bound dihydrotestosterone is achieved through a receptor mechanism or

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“…It was shown that dihydrotestosteronecytosol receptor complex was translocated into nuclei where the complex interacted with chromatin in the rat prostate (Mainwaring and Peterken, 1971;Doorn et al, 1976 ;Davies et al, 1976). In nuclei of the rat prostate a limited part seems to be responsible for binding with the hormonereceptor complex (Mainwaring et al, 1976a, b).…”

Section: Discussionmentioning

confidence: 99%