Markaverich B M, Gregory R R
Center for Biotechnology, Baylor College of Medicine, Woodlands, TX 77381.
Steroids. 1993 Jun;58(6):268-74. doi: 10.1016/0039-128x(93)90071-t.
Naturally occurring bioflavonoids such as luteolin compete for [3H]estradiol binding to nuclear type II sites and mimic methyl p-hydroxyphenyllactate (MeHPLA) as ligands for this cell regulatory protein. More importantly, luteolin (3',4',5,7-tetrahydroxyflavone) contains catechol 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. The preliminary experiments presented in this manuscript demonstrate that luteolin and a related bioflavonoid, 4,7-dihydroxyflavone (DHF), are competitive inhibitors of [3H]estradiol binding to type II sites in ammonium sulfate (AmSO4) extracts of rat uterine nuclei. This high affinity (Kd 5-10 nM) interaction is specific for type II sites, and neither compound binds to the estrogen receptor (ER). More importantly, the interaction of luteolin with nuclear type II sites was irreversible, whereas DHF readily exchanged with [3H]estradiol for type II sites in these preparations. These findings suggest that this nonexchangable occupancy of type II sites by luteolin is likely to involve covalent attachment. Spectrophotometric analysis of type II site preparations pretreated with luteolin also confirmed the [3H]estradiol exchange assay data, demonstrating that the ligand attachment is irreversible. Because luteolin did not affect [3H]estradiol binding to the ER in uterine cytosol, we suspect that this bioflavonoid may not be simply randomly interacting with a multiplicity of proteins to generate covalent complexes. These preliminary findings suggest that high-affinity binding of luteolin by type II sites is prerequisite to covalent attachment and that this bioflavonoid may be a suitable affinity ligand for the purification of this protein.
天然存在的生物类黄酮，如木犀草素，可竞争[3H]雌二醇与核II型位点的结合，并模拟对羟基苯乳酸甲酯（MeHPLA）作为这种细胞调节蛋白的配体。更重要的是，木犀草素（3'，4'，5，7-四羟基黄酮）在A环和B环上含有儿茶酚羟基，这些羟基可能形成能够与蛋白质共价结合的醌；因此，我们评估了木犀草素作为大鼠子宫核II型位点潜在亲和配体的可能性。本手稿中呈现的初步实验表明，木犀草素和一种相关的生物类黄酮4,7-二羟基黄酮（DHF）是[3H]雌二醇与大鼠子宫核硫酸铵（AmSO4）提取物中II型位点结合的竞争性抑制剂。这种高亲和力（Kd为5-10 nM）的相互作用对II型位点具有特异性，且这两种化合物均不与雌激素受体（ER）结合。更重要的是，木犀草素与核II型位点的相互作用是不可逆的，而在这些制剂中，DHF很容易与[3H]雌二醇交换II型位点。这些发现表明，木犀草素对II型位点的这种不可交换占据可能涉及共价连接。对用木犀草素预处理的II型位点制剂进行分光光度分析也证实了[3H]雌二醇交换试验数据，表明配体连接是不可逆的。由于木犀草素不影响[3H]雌二醇与子宫胞质溶胶中ER的结合，我们怀疑这种生物类黄酮可能不是简单地随机与多种蛋白质相互作用以生成共价复合物。这些初步发现表明，II型位点对木犀草素的高亲和力结合是共价连接的先决条件，并且这种生物类黄酮可能是纯化该蛋白质的合适亲和配体。
