Wiedenmann B, Lawley K, Grund C, Branton D
J Cell Biol. 1985 Jul;101(1):12-8. doi: 10.1083/jcb.101.1.12.
To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.
为了鉴定整合膜蛋白和外周膜蛋白，将来自牛脑的高度纯化的包被小泡暴露于具有不同pH值、离子强度和非离子去污剂Triton X-100浓度的溶液中。在pH 10.0或更高时，大多数主要蛋白质被释放出来，但有四种次要多肽与小泡一起沉淀。通过对沉淀和提取物中磷脂的定量分析，我们确定在pH高达12时，所有磷脂都可以在沉淀中回收。在pH 12.0下对包被小泡进行电子显微镜检查，发现所有小泡都没有包被结构。在pH 6.5 - 8.5下用高离子强度溶液（0 - 1.0 M KCl）处理也能释放所有主要蛋白质，除了微管蛋白，它仍然可沉淀。向包被小泡或去除了90%网格蛋白的剥离小泡中添加Triton X-100，会释放出四种不同的多肽，其分子量约为38,000、29,000、24,000和10,000。分子量为38,000的多肽（pK约为5.0），约占Triton X-100释放的蛋白质的50%，基于其与过碘酸-希夫试剂的反应，似乎是一种糖蛋白。先提取90%的网格蛋白，然后用Triton X-100提取90%的磷脂，产生了一个仍然可沉淀的蛋白质残余物，其由似乎是收缩的剥离小泡的结构组成。我们的数据共同表明，脑包被小泡的大多数主要多肽表现为外周膜蛋白，至少有四种多肽表现为整合膜蛋白。通过使用单克隆抗体，我们已将其中一种多肽（分子量38,000）鉴定为小牛脑包被小泡亚群的标志物。
