Identification of nuclear type II [(3)H]estradiol binding sites as histone H4.
[3H]Luteolin binds covalently to uterine nuclear type II sites [B. Markaverich, K. Shoulars, M.A. Alejandro, T. Brown, Steroids 66 (2001) 707] and was used to identify this protein(s). SDS-PAGE analyses of [3H]luteolin-labeled type II site preparations revealed specific binding to 11- and 35-kDa proteins. The 11-kDa protein was identified as histone H4 by amino acid sequencing. Western blotting confirmed that the 11- and 35-kDa proteins were acetylated forms of histone H4. Anti-histone H4 antibodies (but not H2A, H2B, or H3 antibodies) quantitatively immunoadsorbed type II binding sites from nuclear extracts. Binding analyses by [3H]estradiol exchange, using luteolin as a competitor, detected specific type II binding activity to histone H4 (but not histones H2A, H2B, or H3) generated in a rabbit reticulocyte lysate translation system and confirmed that histone H4 is the type II site.
Shoulars
Kevin
K
Department of Molecular and Cellular Biology and Center for Comparative Medicine, Baylor College of Medicine, One Baylor Plaza, 77030, Houston, TX, USA.
Brown
Trellis
T
Alejandro
Mary Ann
MA
Crowley
Jan
J
Markaverich
Barry M
BM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D005419:Flavonoids / Q000378:metabolism; D005471:Fluoroscopy; D006657:Histones / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007163:Immunosorbent Techniques; D007773:Lactates / Q000378:metabolism; D047311:Luteolin; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D020539:Sequence Analysis, Protein; D013950:Thymus Gland / Q000737:chemistry; D014316:Tritium
D000906:Antibodies; D005419:Flavonoids; D006657:Histones; D007773:Lactates; D014316:Tritium; D004958:Estradiol; C055915:methyl 4-hydroxyphenyllactate; D001120:Arginine; D047311:Luteolin