A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.
A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.
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DOI：
10.1083/jcb.109.4.1753
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发表时间：
1989-10
影响因子：
7.8
通讯作者：
Cohen, J B
中科院分区：
文献类型：
--
作者：
Carr, C;Fischbach, G D;Cohen, J B
为了鉴定与烟碱突触后膜相关的蛋白质，制备了电鳐电器官富含乙酰胆碱受体（AChR）的膜蛋白的单克隆抗体。获得的抗体识别两种新的蛋白质，87，000 Mr和210，000：220，000的双联体，以及先前描述的蛋白质，43，000 Mr，58，000（在我们的凝胶系统中为51，000），270，000和37，000（calelectin）。87-kD的蛋白copurified与乙酰胆碱受体和43-和51-kD的蛋白质在平衡离心过程中连续蔗糖梯度，而大部分的210/220-kD的蛋白质从乙酰胆碱受体分离。87-kD蛋白仍然与受体和43-kD蛋白在速度沉降过程中通过浅蔗糖梯度，一个程序，分离出大量的51-kD蛋白AChRs。87-和270-kD的蛋白质被切割的Ca++激活的蛋白酶存在于粗制剂，也在高度纯化的突触后膜。与抗37 kD抗体的例外，一些单克隆抗体对鱼雷蛋白也承认鸡和/或大鼠骨骼肌纤维的冷冻切片和透化鸡肌管在体外生长的决定因素。抗87-kD位点集中在鸡和大鼠终板，但抗体也认识到存在于较低的网站密度在突触外膜的决定因素。抗210：220-kD标记的小鸡终板，但神经元-肌管共培养的研究表明，这种抗原位于神经突，而不是突触后膜。在其他物种中，43-kD决定簇仅限于鸡终板和抗-51-kD和抗-270-kD标记的突触外以及突触膜。没有交叉反应抗体识别完整（未透化）肌管上的决定簇，因此抗原必须位于表面膜的细胞质方面。每个细胞内的决定因素在AChR固定在发展和成熟终板的作用仍有待研究。
To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++- activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.