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.
Department of Molecular and Cellular Biology and Center for Comparative Medicine, Baylor College of Medicine, One Baylor Plaza, 77030, Houston, TX, USA.
Kevin
K
Shoulars
Trellis
T
Brown
Mary Ann
MA
Alejandro
Jan
J
Crowley
Barry M
BM
Markaverich
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