Böni-Schnetzler M, Scott W, Waugh S M, DiBella E, Pilch P F
J Biol Chem. 1987 Jun 15;262(17):8395-401.
Treatment of the soluble insulin receptor from human placenta with 1.25 mM dithiothreitol and 75 mM Tris at pH 8.5 results in complete reduction of interhalf disulfide bonds (class 1 disulfides) and dissociation of the tetrameric receptor into the dimeric alpha beta form. The alpha beta receptor halves exhibit a reduced affinity for insulin binding (Böni-Schnetzler, M., Rubin, J. B., and Pilch, P. F. (1986) J. Biol. Chem. 261, 15281-15287). Kinetic experiments reveal that reduction of class 1 disulfides is a faster process than the loss of affinity for ligand, indicating that events subsequent to reduction of interhalf disulfides are responsible for the affinity change. We show that a third class of alpha subunit intrachain disulfides is more susceptible to reduction at pH 7.6 than at pH 8.5 and appears to form part of the ligand binding domain. Reduction of the intrachain disulfide bonds in this part of the alpha subunit leads to a loss of insulin binding. Modification of this putative binding domain by dithiothreitol can be minimized if reduction is carried out at pH 8.5. When the insulin receptor in placental membranes is reduced at pH 8.5, the receptor's affinity for insulin is not changed when binding is measured in the membrane. However, the Kd for insulin binding is reduced 10-fold when alpha beta receptor halves are subsequently solubilized. Scatchard analysis of insulin binding to reduced or intact receptors in the membrane and in soluble form together with sucrose density gradient analysis of soluble receptors suggests that alpha beta receptor halves remain associated in the membrane after reduction, but they are dissociated upon solubilization. We interpret these results to mean that the association of two ligand binding domains, 2 alpha beta receptor halves, is required for the formation of an insulin receptor with high affinity for ligand.
在pH 8.5条件下，用1.25 mM二硫苏糖醇和75 mM Tris处理人胎盘可溶性胰岛素受体，可使链间二硫键（1类二硫键）完全还原，四聚体受体解离为二聚体αβ形式。αβ受体亚基对胰岛素结合的亲和力降低（博尼 - 施内茨勒，M.，鲁宾，J. B.，和皮尔希，P. F.（1986年）《生物化学杂志》261，15281 - 15287）。动力学实验表明，1类二硫键的还原过程比配体亲和力丧失的过程更快，这表明链间二硫键还原后的后续事件是亲和力变化的原因。我们发现，α亚基链内的第三类二硫键在pH 7.6时比在pH 8.5时更容易被还原，并且似乎构成配体结合结构域的一部分。α亚基这部分的链内二硫键还原会导致胰岛素结合丧失。如果在pH 8.5进行还原，二硫苏糖醇对这个假定结合结构域的修饰可以最小化。当胎盘膜中的胰岛素受体在pH 8.5下被还原时，在膜中测量结合时，受体对胰岛素的亲和力没有变化。然而，当随后将αβ受体亚基溶解时，胰岛素结合的解离常数降低了10倍。对膜中及可溶形式的还原或完整受体进行胰岛素结合的斯卡查德分析，以及对可溶受体进行蔗糖密度梯度分析表明，还原后αβ受体亚基在膜中仍保持结合状态，但溶解后会解离。我们将这些结果解释为，两个配体结合结构域（2个αβ受体亚基）的结合是形成对配体具有高亲和力的胰岛素受体所必需的。
