Steroid binding to hepatoma tissue culture cell glucocorticoid receptors involves at least two sulfhydryl groups.

Differential effects of the reversible thiol-reactive agents arsenite and methyl methanethiosulfonate on steroid binding by the glucocorticoid receptor

“…The supernatant from this centrifugation was removed and is referred to as "cytosol". HTC cell cytosol was prepared by freeze-thaw lysis of cells grown in spinner culture in S77 medium supplemented with 5% fetal and 5% newborn bovine sera (Biofluids) and 0.03% glutamine as described by Miller and Simons (1988).…”

“…To understand the molecular mechanism of glucocorticoid hormone action, we must define those features of glucocorticoid receptor (GR)1 secondary and tertiary structure that determine the high-affinity steroid binding conformation. The most important of these determinants defined to date are the presence or absence of an intramolecular disulfide for a vicinally spaced pair of cysteine SH groups lying in the GR steroid binding domain (Miller & Simons, 1988;) and the association of the GR with hsp90 (Bresnick et al, 1989;Scherrer et al, 1990).…”

“…1 Abbreviations: GR, glucocorticoid receptor; hsp90, the 90-kDa heat shock protein; triamcinolone acetonide, 9a-fluoro-ll/3,16a,17a,21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide; dexamethasone, 9a-fluoro-16a-methyl-11/3,17a,21 -trihydroxypregna-1,4-diene-3,20- 0006-2960/93/0432-3729S04.00/0 Bodwell et al (1984) first introduced the use of the reversible reagent methyl methanethiosulfonate (MMTS) for the study of receptor SH groups. MMTS treatment reduces the affinity of the GR for the traditional agonists dexamethasone and cortisol by about a factor of 5, while there is very little, if any, decrease in the affinity for the nonconventional steroids deacylcortivazol and RU38486 (Miller & Simons, 1988). MMTS reacts in a highly specific manner with protein thiols to derivatize them by adding a thiomethyl group through formation of a mixded disulfide (Brocklehurst, 1979).…”

“…The SH group can be regenerated by reagents that promote thioldisulfide interchange. At low concentrations, MMTS appears to promote the formation of an intramolecular disulfide bond in the receptor (Miller & Simons, 1988; Chakraborti et al, 1990, implying that two thiol groups must be located very close to each other in the properly folded high-affinity steroid binding conformation of the hormone binding domain.…”

Αλληλεπιδραση Του Υποδοχεα Γλυκοκορτικοειδων Με Τις Πρωτεινες Θερμικου Σοκ

“…Το μολυβδαινικό νάτριο διατηρεί παράλληλα το μη μετασχηματισμένο υποδοχέα ενεργό, δηλαδή σε μορφή ικανή να συνδέει ορμόνη με υψηλή χημική συγγένεια. Η απενεργοποίηση του μη μετασχηματισμένου υποδοχέα, δηλαδή η απώλεια της ικανότητας σύνδεσης ορμόνης, φαίνεται να συνδυάζεται με αποφωσφορυλίωση (Nielsen et al, 1977) και πιθανά οφείλεται στην οξείδωση σουλφυδρυλομάδων του ενεργού κέντρου σύνδεσης της ορμόνης (Miller and Simons, 1988). Παρουσία μολυβδαινικού νατρίου η ανενεργοποίηση είναι πλήρως αντιστρέψιμη μετά από προσθήκη DTT (Housley et al, 1982).…”