Showing papers by "Allan Munck published in 1988"

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

Abstract: Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate(ABSTRACT TRUNCATED AT 250 WORDS)