Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA

O. Wrange, J. Carlstedt-Duke, J. A. Gustafsson

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37 Scopus citations

Abstract

Purified preparations of activated glucocorticoid·receptor complex (GR) contain a M(r) 94,000 hormone-binding polypeptide co-purifying together with a M(r) 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt-Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G. L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J.-A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection 'footprinting' analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate M(r) 94,000 binding to specific and nonspecific DNA sites. One M(r) 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of M(r) 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR·DNA complex contained similar amounts of M(r) 94,000 and M(r) 72,000 polypeptide. It is as yet uncertain if the M(r) 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.

Original languageEnglish (US)
Pages (from-to)11770-11778
Number of pages9
JournalJournal of Biological Chemistry
Volume261
Issue number25
StatePublished - 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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