J. Biol. Chem., Vol. 262, Issue 12, 5530-5535, Apr, 1987
Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits
G Redeuilh, C Secco, J Mester and EE Baulieu
The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S
receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in
the presence of either an excess of unlabeled hormone ("chase") or of
charcoal/dextran suspension ("stripping"). Biphasic dissociation of the
hormone was observed in both cases, but the fraction of the
fast-dissociating component was dramatically reduced (5% instead of 60%)
when stripping was used. As the dissociation patterns were independent of
the degree of saturation of the receptor, the results do not favor the
possibility of cooperative effects between binding sites in the 8-9 S ER.
After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the
dissociation studied by chase displayed only the slowly dissociating
component (t1/2 approximately 65 min). This effect was dependent on
temperature and influenced by the ligand bound to 8-9 S ER, being
pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The
slow-dissociating component obtained after charcoal treatment was
reconverted to fast-dissociating state by adding dithiothreitol or by
incubation with cytosol at 20 degrees C. The charcoal treatment did not
change the sedimentation coefficient (approximately 9 S) and the Stokes
radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the
slow-dissociating form obtained did not bind to DNA-cellulose either in the
presence or absence of molybdate ions. Thus there are likely small but
functionally significant changes of structure in the 8-9 S ER which remain
in a non- DNA-binding form, whereas the rate of estradiol dissociation is
modified.
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