J. Biol. Chem., Vol. 260, Issue 18, 10093-10098, Aug, 1985
In situ cross-linking of androgen receptors to nuclear acceptor sites of rat prostate with formaldehyde
JA Foekens, PS Rennie, H Cheng and N Bruchovsky
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.
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