Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein.
Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein.
复制标题
用纯化的猪心房抑制性鸟嘌呤核苷酸结合蛋白重建纯化的猪心房毒蕈碱乙酰胆碱受体。
DOI：
10.1021/bi00399a023
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Schimerlik,MI
中科院分区：
文献类型：
--
作者：
Tota,MR;Kahler,KR;Schimerlik,MI
Oregon State University，Corvallis，Oregoti 97331生物化学和生物物理系，1987年6月10日收到修订稿件，1987年8月18日收到修订稿件摘要：纯化的猪心房M受体(MAcChR)与纯化的猪心房抑制鸟氨酸核苷酸结合蛋白(GJ)在由磷脂酰胆碱、磷脂酰丝氨酸和胆固醇(1：1：0.1w/w)组成的脂质混合物中重组。5‘-鸟氨酰亚胺二磷酸(0.1 mM)对毒扁豆碱拮抗剂L-奎宁基苯甲酸酯的结合无影响，但使重组制剂中高亲和力的卡巴胆碱结合部位(Kd=1 pm)转变为低亲和力状态(Kd=约100 pm)。稳态动力学测定表明，在M受体拮抗剂L的存在下，GTP酶的转化率从0.19min-1增加到激动剂卡巴胆碱的2.11min-1。与卡巴胆碱-mAcChR络合物相互作用后，GJ对GDP的亲和力降低了约50倍，观察到的GDP解离速率常数从0.12增加到4.5min-1，增加了38倍。因此，在M受体激动剂中观察到的稳态GTPase活性的增加很大程度上(如果不是完全的话)是由于GDP从G解离的增加--可能是稳态机制中的限速步骤。氨甲酰胆碱刺激的GTP酶对百日咳毒素对重组G_1的ADP-核糖化反应敏感，但高亲和力激动剂的结合只有在百日咳毒素和激动剂乙酰胆碱存在的情况下才能解偶联。这些结果表明，与mAcChR的结合可保护G_1免受百日咳毒素的ADP-核糖化。乙酰胆碱受体(MAcChR)1属于神经递质受体的一类，其信号转导由鸟嘌呤核苷酸结合调节蛋白介导。刺激腺酰环化酶的受体已被证明通过Gs相互作用，而抑制该酶的受体，如mAcChR，似乎通过G偶联(Stryer&Bourne，1986)。在心房组织中，被mAcChRs激活的G也被证明是一种
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregoti 97331 Received June 10, 1987; Revised Manuscript Received August 18, 1987 abstract: Purified porcine atrial muscarinic receptor (mAcChR) was reconstituted with purified porcine atrial inhibitory guanine nucleotide binding protein (Gj) in a lipid mixture consisting of phosphatidylcholine, phosphatidylserine, and cholesterol (1: 1: 0.1 w/w). 5'-Guanylyl imidodiphosphate (0.1 mM) had no effect on the binding of the muscarinic antagonist L-quinuclidinyl benzilate but converted high-affinity carbachol binding sites {Kd equal to 1 pM) in the reconstituted preparation to the low-affinity state (Kd equal to about 100 pM). Steady-state kinetic measurements of GTPase activity showed that the turnover number was increased from 0.19 min-1 in the presence of the muscarinic antagonist L-hyoscyamine to 2.11 min-1 for the agonist carbachol. The affinity of Gj for GDP was reduced by about 50-fold upon interaction with the carbachol-mAcChR complex, and the observed rate constant for GDP dissociation was increased by 38-fold from 0.12 to 4.5 min-1. Thus, the increase in steady-state GTPase activity observed for muscarinicagonists is largely, if not exclusively, due to the increase in GDP dissociation from G;—probably the rate-limiting step in the steady-statemechanism. Carbachol-stimulated GTPase was sensitive to ADP-ribosylation of the reconstituted G; by pertussis toxin, but the high-affinity agonist binding was uncoupled only when the reconstituted preparation was treated with pertussis toxin in the presence of GTP and the agonist acetylcholine. These results suggest that association with the mAcChR protects G; fromADP-ribosylation bypertussis toxin. e muscarinic acetylcholine receptor (mAcChR) 1 belongs to the class of neurotransmitter receptors in which signal transduction is mediated by guanine nucleotide binding reg-ulatory proteins. Receptors that stimulate adenylyl cyclase have been shown to interact through Gs, while receptors that inhibit the enzyme, such as the mAcChR, appear to be coupled through G;(Stryer & Bourne, 1986). In atrial tissue, G, activated by mAcChRs has also been shown to function as a