DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate

Paul S. Rennie, Jean Francois Bowden, Nicholas Bruchovsky, Paul S. Frenette, John A. Foekens, Helen Cheng

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17β-succinyl agarose, eluted with 75 μM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone- 17β-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-Iabelled proteins-one corresponding to a ptotein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.

Original languageEnglish (US)
Pages (from-to)513-520
Number of pages8
JournalJournal of Steroid Biochemistry
Volume27
Issue number1-3
DOIs
StatePublished - 1987
Externally publishedYes

Fingerprint

Androgen Receptors
Nuclear Proteins
Rats
Prostate
DNA
Proteins
Chromatin
Dihydrotestosterone
Affinity chromatography
Affinity Chromatography
Sepharose
Trypsin
Formaldehyde
Molecular Weight
Molecular weight
Endopeptidase K
Halogenation
Cytoplasmic and Nuclear Receptors
Chloroform
Phenol

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate. / Rennie, Paul S.; Bowden, Jean Francois; Bruchovsky, Nicholas; Frenette, Paul S.; Foekens, John A.; Cheng, Helen.

In: Journal of Steroid Biochemistry, Vol. 27, No. 1-3, 1987, p. 513-520.

Research output: Contribution to journalArticle

Rennie, Paul S. ; Bowden, Jean Francois ; Bruchovsky, Nicholas ; Frenette, Paul S. ; Foekens, John A. ; Cheng, Helen. / DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate. In: Journal of Steroid Biochemistry. 1987 ; Vol. 27, No. 1-3. pp. 513-520.
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