Abstract
Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 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 Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 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 X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.
Highlights
Purified preparations of activated glucocorti- ulated enhancers when fused to promoters and transcoid*receptor complex (GR) contain a M, 94,000 fected [16,17,18]
Glycerol den- entity with M, of about 94,000 as studiedby SDS-PAGE (22, sity gradient centrifugation was used to quantitate M, 24, 26)
Glycerol gradient centrifugation fol- terms of (i) it does not bind hormone; (ii) it does not react lowed bysodium dodecyl sulfate-polyacrylamide gel with antibodies raised against the M, 94,000 entity; (iii) itis electrophoresis showed that the specific GR*DNAcomplex contained similar amounts of M, 94,000 and M, 72,000 polypeptide
Summary
Reference band to normalize for variations in DNaseI digestion. The bands were cut out and counted in the liquid scintillation counter in order to quantitate theGR.DNA binding. Parallel gradients containingonly end-labeled DNA before incubation a t 25 "C. or only purified receptor were always present. For experiments where the fractions after gradient centrifugation were to be analyzed by SDS-PAGE, 200 pl of purified receptor Samples from the purified receptor preparations (4 pg of GR from the pool after DEAE-Sepharose) were stained using Coomassie Brillant Blue and analyzed by densitometry a t 550 nm in a Beckman R-. Purified GR and DNA in each of the peaks on the glycerol gradients was quanti- from three separate purifications [1,2,3] was analyzed by SDS-PAGE tated from the amount of 3H and 32Pradioactivity, respectively.
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