The specificity of
protein labeling by an affinity label of
glucocorticoid receptors,
dexamethasone 21-mesylate (Dex-Mes), was investigated using
bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized
cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the
thiol-specific
reagent methyl methanethiolsulfonate (
MMTS). Thus both Dex-Mes and
MMTS appear to react very selectively with
thiols under our conditions. In reactions with
hepatoma tissue culture (HTC) cell
glucocorticoid receptors,
MMTS was equally efficient in preventing [3H]
dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor
protein. These results indicate that Dex-Mes labeling of the
glucocorticoid receptor involves covalent reaction with at least one
cysteine in the
steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with
trypsin,
chymotrypsin, and Staphylococcus aureus V8
protease under denaturing conditions. Data from these fragments on 15%
sodium dodecyl sulfate-
polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit
protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with
trypsin,
chymotrypsin, or V8
protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the
amino acids immediately surrounding the affinity-labeled
cysteine(s) of the
steroid binding site.