Wang S L, Raizada M K, Shiverick K T
Department of Pharmacology and Experimental Therapeutics, University of Florida, Gainesville 32610.
Mol Pharmacol. 1988 Mar;33(3):250-6.
Studies investigating the effects of beta-naphthoflavone (beta NF) on insulin receptor binding and its intrinsic protein kinase activity in rat liver and placenta were performed. Membranes were prepared from maternal liver and placenta on gestation day 11 and used for [125I]insulin radioreceptor assay. Scatchard analysis showed that association constants (Ka) for high affinity binding sites were similar for placental and liver membrane. The administration of beta NF, 15 mg/kg, 1 day before study did not alter the specific binding of insulin to liver membranes, whereas ligand binding to placental preparations was decreased 40% from control. Scatchard analysis of binding to placental membranes suggests that beta NF treatment was associated with a change in the number of high affinity binding sites. In further studies membrane receptors were solubilized and partially purified by wheat germ agglutinin affinity chromatography for protein kinase assay. Insulin stimulated the phosphorylation of the Mr 95,000 subunit of the receptor in lectin-purified membrane proteins from liver and placenta. In liver receptor preparations, beta NF treatment was associated with a nearly 3-fold increase in the insulin-stimulated phosphorylation of the 95-kD protein. In contrast, placental receptor preparations showed a 40% decrease in the extent of autophosphorylation following beta NF treatment. Insulin-stimulated phosphorylation of an exogenous substrate poly(Glu4, Tyr) also showed a divergent pattern of changes in liver and placental receptors following beta NF treatment. In studies during late gestation (day 18), beta NF treatment was also associated with an increase in liver receptor kinase activity, whereas placental receptors showed a decrease in autophosphorylation. Thus, acute treatment with beta NF during mid and late gestation was associated with significant alterations in insulin receptor protein kinase activity, and data suggest that fetal insulin receptors may respond in a different manner than maternal receptors to polyaromatic compounds like beta NF. The observed effects of beta NF on liver and placental receptor kinase activity may be related to alterations in insulin function in the regulation of pregnancy and fetoplacental growth.
开展了多项研究，以调查β-萘黄酮（β-NF）对大鼠肝脏和胎盘胰岛素受体结合及其内在蛋白激酶活性的影响。在妊娠第11天从母体肝脏和胎盘制备细胞膜，并用于[125I]胰岛素放射受体分析。Scatchard分析表明，胎盘和肝细胞膜高亲和力结合位点的结合常数（Ka）相似。在研究前1天给予15mg/kg的β-NF，并未改变胰岛素与肝细胞膜的特异性结合，而与胎盘制剂的配体结合较对照降低了40%。对胎盘膜结合的Scatchard分析表明，β-NF处理与高亲和力结合位点数量的变化有关。在进一步的研究中，通过麦胚凝集素亲和层析将膜受体溶解并部分纯化，用于蛋白激酶分析。胰岛素刺激肝脏和胎盘凝集素纯化膜蛋白中受体95,000 Mr亚基的磷酸化。在肝脏受体制剂中，β-NF处理使95-kD蛋白的胰岛素刺激磷酸化增加近3倍。相反，胎盘受体制剂在β-NF处理后自磷酸化程度降低40%。胰岛素刺激的外源底物聚（Glu4，Tyr）磷酸化在β-NF处理后肝脏和胎盘受体中也呈现出不同变化模式。在妊娠晚期（第18天）的研究中，β-NF处理也与肝脏受体激酶活性增加有关，而胎盘受体自磷酸化降低。因此，在妊娠中期和晚期用β-NF急性处理与胰岛素受体蛋白激酶活性的显著改变有关，数据表明胎儿胰岛素受体对β-NF等多环芳烃化合物的反应可能与母体受体不同。观察到的β-NF对肝脏和胎盘受体激酶活性的影响可能与胰岛素在调节妊娠和胎儿胎盘生长中的功能改变有关。
