Turyn D, Scacchi G E, Dellacha J M
Biochim Biophys Acta. 1985 Jun 30;845(3):333-42. doi: 10.1016/0167-4889(85)90196-x.
The specific binding of [125I]insulin to submaxillary gland microsomes was significantly enhanced by increasing the ionic strength of the incubation medium. This effect was neither related to changes in receptor or hormone degradation nor in the polymerization of the tracer. When equilibrium binding data from competition curves of unlabelled insulin versus [125I]insulin were analyzed, a marked increase in total binding capacity in high ionic strength was observed (from 890 to 2440 fmol/mg protein), with no change in binding affinity. Phospholipase C digestion was also able to increase specific [125I]insulin binding to microsomes. These results suggest the presence of masked receptors in submaxillary gland microsomes. Methylation of rat submaxillary gland microsomes by using S-adenosyl-L-methionine as the methyl donor significantly increased [125I]insulin binding. Scatchard analysis of the equilibrium binding data showed that addition of S-adenosyl-L-methionine (0.46 mM) to microsomes resulted in an enhancement of the total binding capacity (from 990 to 1520 fmol/mg protein) with no change in the affinity constants, which suggests the exposure of masked insulin receptors under such conditions. Both the methyl group incorporation into membrane phospholipids and the effect on insulin binding were dependent on the S-adenosyl-L-methionine concentration used and were partially suppressed in the presence of S-adenosyl-L-homocysteine, a specific competitive inhibitor of the methyltransferases activity. When microsomes were treated with S-adenosyl-L-[methyl-3H]methionine, the 3H-labelled methyl groups incorporated were found mainly in the lipid fraction associated to phosphatidylcholine, suggesting in this case that the unmasking of insulin receptors could be a consequence of alterations produced in membrane composition. The effects of phospholipase C, S-adenosyl-L-methionine and high ionic strength on insulin binding were not additive, suggesting that these procedures unmask receptors from the same pool.
通过增加孵育介质的离子强度，[125I]胰岛素与颌下腺微粒体的特异性结合显著增强。这种效应既与受体或激素降解的变化无关，也与示踪剂的聚合无关。当分析未标记胰岛素与[125I]胰岛素竞争曲线的平衡结合数据时，观察到在高离子强度下总结合能力显著增加（从890增加到2440 fmol/mg蛋白质），而结合亲和力没有变化。磷脂酶C消化也能够增加[125I]胰岛素与微粒体的特异性结合。这些结果表明颌下腺微粒体中存在隐蔽受体。以S-腺苷-L-甲硫氨酸作为甲基供体对大鼠颌下腺微粒体进行甲基化，可显著增加[125I]胰岛素结合。对平衡结合数据的Scatchard分析表明，向微粒体中添加S-腺苷-L-甲硫氨酸（0.46 mM）会导致总结合能力增强（从990增加到1520 fmol/mg蛋白质），而亲和力常数没有变化，这表明在这种条件下隐蔽的胰岛素受体被暴露。甲基掺入膜磷脂以及对胰岛素结合的影响均取决于所使用的S-腺苷-L-甲硫氨酸浓度，并且在甲基转移酶活性的特异性竞争性抑制剂S-腺苷-L-高半胱氨酸存在的情况下会部分受到抑制。当用S-腺苷-L-[甲基-3H]甲硫氨酸处理微粒体时，发现掺入的3H标记甲基主要存在于与磷脂酰胆碱相关的脂质部分，在这种情况下表明胰岛素受体的去隐蔽可能是膜组成发生改变的结果。磷脂酶C、S-腺苷-L-甲硫氨酸和高离子强度对胰岛素结合的影响不是累加的，这表明这些方法从同一组中去隐蔽受体。
