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

Journal Article · Tue May 17 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00410a034· OSTI ID:7191973

Smith, L I; Bodwell, J E; Mendel, D B; Ciardelli, T; North, W G; Munck, A

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors 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 (/sup 3/H)dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single (/sup 3/H)dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the (/sup 3/H)dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with (/sup 3/H)dexamethasone 21-mesylate and (/sup 35/S)methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of (/sup 3/H)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, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells.

• Research Organization: Dartmouth Medical School, Hanover, NH (USA)
• OSTI ID: 7191973
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:10; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADRENAL HORMONES
AMINO ACID SEQUENCE
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHROMATOGRAPHY
CONFIGURATION INTERACTION
CORTICOSTEROIDS
CYSTEINE
DAYS LIVING RADIOISOTOPES
DEXAMETHASONE
DRUGS
DUAL-ISOTOPE SUBTRACTION TECHNIQUE
ENZYMES
EVEN-ODD NUCLEI
GLUCOCORTICOIDS
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KETONES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
LIQUID COLUMN CHROMATOGRAPHY
MEMBRANE PROTEINS
METHIONINE
MOLECULAR STRUCTURE
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HYDROLASES
PREGNANES
PROTEINS
RADIOISOTOPES
RECEPTORS
SEPARATION PROCESSES
SERINE PROTEINASES
STEROIDS
SULFUR 35
SULFUR ISOTOPES
THIOLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TRYPSIN