Purification and characterization of nuclear type II [(3)H]estradiol binding sites from the rat uterus: covalent labeling with [(3)H]luteolin.
Type II [3H]estradiol binding sites play an important role in normal and malignant cell growth and proliferation and the delineation of the precise function of the type II site in cell growth has been hampered by the inability to purify, sequence and or clone this protein. The present manuscript describes methodology for the solubilization, purification and tentative identification of type II sites from the estrogen-treated rat uterus. This protein(s) chromatographs as a single major peak on DNA-cellulose, Affigel Blue dye affinity resin and during high performance liquid chromatography (HPLC) on hydroxyapatite. The purified fractions from these columns elicited classical [3H]estradiol binding characteristics (sigmoidal saturation curve, hyperbolic Scatchard plot, and Hill coefficient of approximately 4) for type II sites that are typically observed in crude or highly purified nuclear fractions or extracts. The type II binding activity also eluted as a single major component from a ligand affinity resin (GT-18-Sepharose) prepared by coupling 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone to epoxy-activated Sepharose (Pharmacia). The molecular weight of the type II site ([3H]estradiol binding activity) under non-denaturing or denaturing conditions was estimated to be approximately 10-15 kDa by gel filtration HPLC. Similarly, nuclear type II sites covalently labeled with the bioflavonoid, [3H]luteolin, migrated in the 10 kDa range on SDS PAGE. Thus, under these various experimental conditions, nuclear type II sites detected by [3H]estradiol or [3H]luteolin labeling techniques displayed little heterogeneity and appear much smaller than ERalpha or ERbeta or other steroid hormone receptors.
Markaverich
B M
BM
Department of Molecular and Cellular Biology and The Center for Comparative Medicine, Baylor College of Medicine, Houston, Texas 77030, USA. barrym@bcm.tmc.edu
Shoulars
K
K
Brown
M A
MA
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D005419:Flavonoids / Q000378:metabolism*; D047311:Luteolin; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism*
D005419:Flavonoids; D011960:Receptors, Estrogen; C107081:estrogen binding site, type II; D004958:Estradiol; D004247:DNA; D047311:Luteolin