Functional reconstitution of beta-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase.
Functional reconstitution of beta-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase.
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β-肾上腺素能受体和腺苷酸环化酶刺激性 GTP 结合蛋白的功能重建。
DOI：
10.1073/pnas.79.23.7228
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发表时间：
1982
影响因子：
11.1
通讯作者：
Ross,EM
中科院分区：
文献类型：
--
作者：
Pedersen,SE;Ross,EM
描述了一种在磷脂囊泡中β-肾上腺素能受体和腺苷酸环化酶的刺激性鸟嘌呤核苷酸结合蛋白（G/F）的功能重建的方法。β-肾上腺素能受体从火鸡红细胞质膜中溶解，并通过加入二肉豆蔻酰磷脂酰胆碱和通过凝胶过滤除去去污剂而重构为磷脂囊泡。该程序恢复了与[125 I]碘羟苯甲基吲哚洛尔和[3 H]二氢烯丙洛尔结合的能力。将纯化的兔肝G/F加入到受体囊泡中，可以被鸟苷5 ′-[3-硫代]三磷酸以低速率稳定地激活，并且在β-肾上腺素能激动剂存在下，这种激活增加高达4倍。这种G/F激活的刺激对β-肾上腺素能受体激动剂具有特异性，并可被β-肾上腺素能受体拮抗剂特异性阻断。刺激与含有活性β-肾上腺素能受体的囊泡的浓度成比例。在最佳条件下，每个受体激活5至6个G/F分子，表明受体对G/F的催化激活被重建。
A procedure for the functional reconstitution of beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. beta-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of beta-adrenergic agonists. This stimulation of the activation of G/F was specific for beta-adrenergic agonists and could be specifically blocked by beta-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active beta-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F by receptor was reconstituted.