Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor.
Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor.
复制标题
乙酰胆碱受体假定的两亲性螺旋位于膜外的证据。
DOI：
10.1021/bi00415a029
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Dwyer,BP
中科院分区：
文献类型：
--
作者：
Dwyer,BP
加州大学化学系，圣地亚哥，拉霍亚，加州92093接收于1987年12月1日;修订的Mandarin pt接收于1988年2月25日摘要：已经获得的证据表明，分别在乙酰胆碱受体α和β亚基的潜在两亲性螺旋中发现的肽GVKYIAE和AIKYIAE并不埋在膜中。合成肽KYIAE，并制备针对牛血清白蛋白和合成肽的缀合物的多克隆抗体。通过将这些特异性抗体连接到琼脂糖上，制备了能够结合并随后释放以序列YIAE结尾的肽的免疫吸附剂。用磷酸吡哆醛和Na [3 H] BH 4标记天然乙酰胆碱受体。将标记的蛋白质剥离磷脂并用来自金黄色葡萄球菌菌株V8的蛋白酶消化。将消化物进行免疫吸附以分离标记的内源肽。作为对照，将通过凝胶过滤乙酰胆碱受体在去污剂中的溶液制备的α和β多肽除去去污剂，并在8 M尿素存在下用磷酸吡哆醛和Na [3 H] BH 4标记。消化标记的α和β多肽并进行免疫吸附。在天然和变性条件下标记的α和β亚基的天然肽的比放射性几乎相等。在使用羟乙基亚硫酰（2/，4/-二硝基苯基）-3-氨基丙酰亚胺酯作为标记剂的类似实验中，来自天然和变性受体的固有肽也被标记到相同程度。因为无论蛋白质是否变性，这些肽的标记程度都是相同的，所以它们不能被埋在膜中。烟碱型乙酰胆碱受体（Iicotinic acetylcholine receptor，IECHR）是研究最多的跨膜蛋白，存在于加利福尼亚电鳐（Torpedo californica）的电器官的突触后膜中，能够形成无机离子通道。在结合乙酰胆碱时，通道打开，允许Na+和K+的净电流以其各自的电化学梯度的方向流过质膜。该受体由四种独特的多肽组成，根据电泳迁移率分别命名为a、y、y和，化学计量为2x2（Reynolds & Karlin，1978）。从图像构建中获得的结构是具有伪5重旋转对称轴的五聚体的结构（Wavesson &
Department of Chemistry, University of California, San Diego, La Jolla, California 92093 Received December 1, 1987; Revised Manuscript Received February 25, 1988 abstract: Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the a and ß subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholinereceptor was labeled with pyridoxal phosphate and Na [3H] BH4. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenouspeptides. As a control, a and ß polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na [3H] BH4 in the presence of 8 M urea. The labeled a and ß polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenouspeptides from the a and ß subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2/, 4/-dinitrophenyl)-3-aminopropionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane.^ íicotinic acetylcholine receptor, found in the postsynaptic membranes of the electric organs of Torpedo californica, is the most widely studied membrane-spanning protein capable of forming a channel for inorganic ions. Upon binding of acetylcholine, the channel opens, allowing net currents of Na+ and K+ to flow across the plasma membrane in the direction of their respective electrochemical gradients. The receptor is constructed of fourunique polypeptides designated a, ß, y, and according to electrophoretic mobility and has the stoichiometry 2ß (Reynolds & Karlin, 1978). The structure obtained from image construction is that of a pentamer with a pseudo-5-fold rotational axis of symmetry (Brisson &