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Author Bresnick, E. H. ♦ Dalman, F. C. ♦ Pratt, W. B.
Source United States Department of Energy Office of Scientific and Technical Information
Content type Text
Language English
Subject Keyword BASIC BIOLOGICAL SCIENCES ♦ GLUCOCORTICOIDS ♦ RADIOASSAY ♦ PROTEINS ♦ MOLECULAR STRUCTURE ♦ RECEPTORS ♦ STOICHIOMETRY ♦ FIBROBLASTS ♦ IODINE 125 ♦ METHIONINE ♦ MICE ♦ MONOCLONAL ANTIBODIES ♦ SULFUR 35 ♦ TRITIUM COMPOUNDS ♦ ADRENAL HORMONES ♦ AMINO ACIDS ♦ ANIMAL CELLS ♦ ANIMALS ♦ ANTIBODIES ♦ BETA DECAY RADIOISOTOPES ♦ BETA-MINUS DECAY RADIOISOTOPES ♦ CARBOXYLIC ACIDS ♦ CONNECTIVE TISSUE CELLS ♦ CORTICOSTEROIDS ♦ DAYS LIVING RADIOISOTOPES ♦ DRUGS ♦ ELECTRON CAPTURE RADIOISOTOPES ♦ EVEN-ODD NUCLEI ♦ HYDROGEN COMPOUNDS ♦ HYDROXY COMPOUNDS ♦ INTERMEDIATE MASS NUCLEI ♦ IODINE ISOTOPES ♦ ISOTOPES ♦ KETONES ♦ LIGHT NUCLEI ♦ LIPOTROPIC FACTORS ♦ MAMMALS ♦ MEMBRANE PROTEINS ♦ NUCLEI ♦ ODD-EVEN NUCLEI ♦ ORGANIC ACIDS ♦ ORGANIC COMPOUNDS ♦ ORGANIC SULFUR COMPOUNDS ♦ PREGNANES ♦ RADIOISOTOPES ♦ RODENTS ♦ SOMATIC CELLS ♦ STEROIDS ♦ SULFUR ISOTOPES ♦ VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques
Abstract The authors have used three methods to measure the stoichiometry of the glucocorticoid receptor and the 90-kDa heat shock protein (hsp90) in L-cell glucocorticoid receptor complexes that were purified by immunoadsorption to protein A-sepharose with an anti-receptor monoclonal antibody, followed by a minimal washing procedure that permits retention of receptor-associated protein. In two of the methods, receptor was quantitated by radioligand binding, and receptor-specific hsp90 was quantitated against a standard curve of purified hsp90, either on Coomassie blue stained SDS gels by laser densitometry or on Western blots by quantitative immunoblotting with {sup 125}I-labeled counterantibody. The stoichiometry values obtained by densitometry and immunoblotting are 7 and 6 mol of hsp90/mol of receptor, respectively. In a third method, which detects total receptor protein rather than just steroid-bound receptor, the ratio of hsp90 to receptor was determined by immunopurifying receptor complexes from ({sup 35}S)methionine-labeled L cells, and the amount of {sup 35}S incorporated into receptor and hsp90 was corrected for the established methionine content of the respective proteins. These observations lead to the conclusion that the untransformed L-cell glucocorticoid receptor exists in cytosol in a much larger heteromeric complex than considered to date. The authors propose that the 9S receptor form that is commonly observed by density gradient centrifugation, and by gel filtration chromatography, must be a core unit containing two hsp90 and one GR which is derived from this larger structure.
ISSN 00062960
Educational Use Research
Learning Resource Type Article
Publisher Date 1990-01-16
Publisher Place United States
Journal Biochemistry
Volume Number 29
Issue Number 2


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