Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.
Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.
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纯化的毒蕈碱受体与磷脂囊泡中重构的纯化的抑制性鸟嘌呤核苷酸调节蛋白的功能相互作用。
DOI：
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发表时间：
1986
影响因子：
4.8
通讯作者：
M. Ui
中科院分区：
文献类型：
--
作者：
H. Kurose;T. Katada;T. Haga;K. Haga;A. Ichiyama;M. Ui
从大鼠脑中纯化了参与腺苷酸环化酶抑制的GTP结合调节蛋白（Ni），并与从猪脑中纯化的毒蕈碱胆碱能受体一起重组到磷脂囊泡中。添加毒蕈碱激动剂卡巴胆碱可刺激重建Ni的鸟苷5 '-O-（3-[35 S]硫代）-三磷酸（[35 S]GTP γ S）结合和GTP水解活性。卡巴胆碱的作用是增加这些活动的Vmax值，但在大多数情况下，Km值也略有增加。卡巴胆碱结合囊泡的Km的数量级相同的刺激GT3。这种结合的亲和力被GTP γ S降低，表明高亲和力受体-Ni复合物在重组囊泡中以GTP依赖性方式形成。镍与NAD和胰岛激活蛋白（IAP），百日咳毒素，孵育引起ADP核糖基化的α-亚基的镍。受体-Ni相互作用的标准，即卡巴胆碱刺激Ni的活性和GTP γ S对卡巴胆碱结合的影响，不再被观察到，当这种IAP处理的Ni，而不是未处理的Ni，被重组成囊泡，虽然IAP处理和未处理的Ni之间没有差异，在没有卡巴胆碱的情况下观察到的基础活性。不，具有与大鼠脑中Ni非常相似的特征的蛋白质，当它们在相同条件下重组成囊泡时，也与毒蕈碱受体偶联。因此，GTP结合蛋白作为IAP催化ADP-核糖基化的底物能够与磷脂囊泡中的毒蕈碱受体功能性地相互作用。
The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the receptor-Ni interaction, i.e. carbachol stimulation of the activities of Ni and the GTP gamma S effect on carbachol binding, were no longer observed, when this IAP-treated Ni, instead of the nontreated Ni, was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated Ni in their basal activities observable without carbachol. No, the protein with a character very similar to Ni in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.