In situ cross-linking of androgen receptors to nuclear acceptor sites of rat prostate with formaldehyde.
Androgen receptors were attached covalently in situ to their nuclear acceptor sites with the contact site cross-linker, formaldehyde. Chromatin, prepared from sonicated nuclei of rat prostate, was labeled by isotope exchange with [3H]dihydrotestosterone and found to contain 19,000 +/- 900 (mean +/- S.E.) salt-extractable androgen receptors/nucleus which sedimented in the 3-4 S region of 7.6-76% (v/v) glycerol gradients and at a density of approximately 1.28-1.35 g/ml in CsCl gradients. After incubation of the chromatin with 0.5% (w/v) formaldehyde for 1 h at 4 degrees C, there was a 90% reduction in the concentration of free androgen receptors and an increase in the density of the androgen binding sites recovered from CsCl gradients. Extensive digestion of the cross-linked chromatin with micrococcal nuclease liberated 18% of the androgen receptors as 3-4 S entities and caused an overall decrease in the density of the receptor-acceptor complexes. Ribonuclease digestions had no effect on the androgen receptors cross-linked to chromatin. Mild digestion of the cross-linked preparations with trypsin, alone or in combination with micrococcal nuclease, resulted in the release of 74% and 97% of the androgen receptors, respectively. Together, these findings imply that two classes of receptor-acceptor complexes are present in prostatic chromatin--one, containing about 20% of the androgen receptors in which the receptors are in direct contact with DNA but not with proteins and the other, containing most of the androgen receptors in which the receptors are adjacent to acceptor proteins but not to DNA.
D000818:Animals; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D013196:Dihydrotestosterone / Q000378:metabolism; D005557:Formaldehyde / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D011467:Prostate / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011944:Receptors, Androgen; D011987:Receptors, Steroid; D013196:Dihydrotestosterone; D005557:Formaldehyde