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Title: Direct stoichiometric evidence that the untransformed M sub r 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex

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 thatmore » 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.« less

Authors:
; ;  [1]
  1. Univ. of Michigan Medical School, Ann Arbor (USA)
Publication Date:
OSTI Identifier:
6700306
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 29:2; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 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

Citation Formats

Bresnick, E H, Dalman, F C, and Pratt, W B. Direct stoichiometric evidence that the untransformed M sub r 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex. United States: N. p., 1990. Web. doi:10.1021/bi00454a028.
Bresnick, E H, Dalman, F C, & Pratt, W B. Direct stoichiometric evidence that the untransformed M sub r 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex. United States. https://doi.org/10.1021/bi00454a028
Bresnick, E H, Dalman, F C, and Pratt, W B. 1990. "Direct stoichiometric evidence that the untransformed M sub r 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex". United States. https://doi.org/10.1021/bi00454a028.
@article{osti_6700306,
title = {Direct stoichiometric evidence that the untransformed M sub r 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex},
author = {Bresnick, E H and Dalman, F C and Pratt, W B},
abstractNote = {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.},
doi = {10.1021/bi00454a028},
url = {https://www.osti.gov/biblio/6700306}, journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 29:2,
place = {United States},
year = {Tue Jan 16 00:00:00 EST 1990},
month = {Tue Jan 16 00:00:00 EST 1990}
}