PRELIMINARY ASSESSMENT OF LUTEOLIN AS AN AFFINITY LIGAND FOR TYPE-II ESTROGEN-BINDING SITES IN RAT UTERINE NUCLEAR EXTRACTS

Naturally occurring bioflavonoids such as luteolin compete for [H-3]es tradiol binding to nuclear type II sites and mimic methyl p-hydroxyphe nyllactate (MeHPLA) as ligands for this cell regulatory protein. More importantly, luteolin (3',4',5,7-tetrahydroxyflavone) contains catecho l hydroxyl groups on the A and B rings that may form quinones capable of binding covalently to proteins; therefore, we evaluated luteolin as a potential affinity ligand for rat uterine nuclear type II sites. Th e preliminary experiments presented in this manuscript demonstrate tha t luteolin and a related bioflavonoid, 4,7-dihydroxyflavone (DHF), are competitive inhibitors of [H-3]estradiol binding to type II sites in ammonium sulfate (AmSO4) extracts of rat uterine nuclei. This high aff inity (Kd 5-10 nM) interaction is specific for type II sites, and neit her compound binds to the estrogen receptor (ER). More importantly, th e interaction of luteolin with nuclear type II sites was irreversible, whereas DHF readily exchanged with [H-3]estradiol for type II sites i n these preparations. These findings suggest that this nonexchangable occupancy of type II sites by luteolin is likely to involve covalent a ttachment. Spectrophotometric analysis of type II site preparations pr etreated with luteolin also confirmed the [H-3]estradiol exchange assa y data, demonstrating that the ligand attachment is irreversible. Beca use luteolin did not affect [H-3]estradiol binding to the ER in uterin e cytosol, we suspect that this bioflavonoid may not be simply randoml y interacting with a multiplicity of proteins to generate covalent com plexes. These preliminary findings suggest that high-affinity binding of luteolin by type II sites is prerequisite to covalent attachment an d that this bioflavonoid may be a suitable affinity ligand for the pur ification of this protein.