ABSENCE OF INTRAMOLECULAR DISULFIDES IN THE STRUCTURE AND FUNCTION OFNATIVE RAT GLUCOCORTICOID RECEPTORS

The presence of intramolecular disulfides in different functional stat es of the native glucocorticoid receptor in the absence of added oxida nts has been examined on nonreducing SDS-polyacrylamide gels. Possible disulfides were trapped by the reaction of thiols in crude receptor s olutions with methyl methanethiolsulfonate or iodoacetamide. The prese nce of diffuse bands at lower molecular weights than either the fully reduced or the thiol-blocked species for both the intact 98-kDa recept or and the 42-kDa chymotryptic fragment was diagnostic of an intramole cular disulfide(s) that had undergone thiol-disulfide rearrangements. However, both the rearrangements and the formation of intramolecular d isulfides were found to occur only with denatured receptors during gel analysis. It appears that the thiols normally complexed with zinc in the zinc fingers may be recruited for disulfide bond formation. Finall y, even when a documented intramolecular disulfide was formed in solut ions of the native protein, thiol-disulfide rearrangements did not occ ur. The tertiary structure of the receptor is thus constituted in a ma nner that not only limits the formation of disulfides but also prevent s the usually facile rearrangements of disulfide bond-containing struc tures to receptor forms that may have greatly reduced activity. Theref ore, although intramolecular disulfide bonds may be of transitory impo rtance, the structural or functional changes of native glucocorticoid receptors that are associated with steroid binding, activation, and di ssociation of heat shock protein 90 neither involve nor require the fo rmation or reduction of stable intramolecular disulfides.