Preparation and characterization of muscarinic-acetylcholine-receptor-enriched membranes from pig atria.
Preparation and characterization of muscarinic-acetylcholine-receptor-enriched membranes from pig atria.
复制标题
猪心房富含毒蕈碱乙酰胆碱受体的膜的制备和表征。
DOI：
10.1042/bj2020475
复制
发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Schimerlik,MI
中科院分区：
文献类型：
--
作者：
Peterson,GL;Schimerlik,MI
建立了一种从猪心房大规模制备膜的方法，该膜富含10-13倍的M胆碱乙酰胆碱受体。该程序包括在含有150 mM-NaClO4和5 mM-EDTA的溶液中进行差速离心法和蔗糖梯度离心法，以最大限度地减少膜聚集。最终的膜制备结合了约1.1pmoL的L-苯甲酸奎宁酯/毫克蛋白质。无论是新鲜组织还是冰冻组织，都得到了类似的结果。从不同的心房区域获得了大致相同的产率(120pmoL的L-苯甲酸奎宁酯部位/100g组织)和比活度。最终制剂在-80℃的蔗糖缓冲溶液中稳定。在电子显微镜下，膜多呈片状或碎片状，部分为闭合的囊泡，在等密度的Percoll梯度上是不均匀的。标记酶研究表明，该受体与质膜标记鸟苷环化酶(颗粒型)和(Na++K+)激活的ATPase平行富含。从单胺氧化酶和葡萄糖-6-磷酸酶活性的分布来看，线粒体外膜和内质网膜存在明显的污染，但该制剂基本上不含肌浆网、线粒体内膜和溶酶体膜。
A procedure was developed for the large scale preparation of membranes from pig atria which are enriched 10-13 fold in the muscarinic acetylcholine receptor. The procedure involved differential centrifugation and sucrose-gradient centrifugation in solutions containing 150 mM-NaClO4 and 5 mM-EDTA to minimize membrane aggregation. The final membrane preparation bound about 1.1 pmol of L-quinuclidinyl benzilate/mg of protein. Comparable results were obtained with either fresh or frozen tissue. About the same yield (120 pmol of L-quinuclidinyl benzilate sites/100 g of tissue) and specific activity of membranes were obtained from different regions of the atria. The final preparation was stable at -80 degrees C in buffered sucrose solutions. The membranes appeared mostly as sheets or fragments and partly as closed vesicles in the electron microscope and were heterogeneous in isopycnic Percoll gradients. Marker enzyme studies showed that the receptor was enriched in parallel with the plasma membrane markers guanylate cyclase (particulate form) and (Na+ + K+)-activated ATPase. Some contamination by mitochondrial outer and endoplasmic reticulum membranes was evident from the distribution of monoamine oxidase and glucose-6-phosphatase activity, but the preparation was largely free of sarcoplasmic reticulum, mitochondrial inner, and lysosomal membranes.