Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions
Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions
复制标题
普瓦泽基立克次体内膜和外膜的分离及其多肽组成的表征
作者：
D. Smith;H. Winkler
普氏立克次体在法国压力细胞中被破坏，并分成可溶性（细胞质）和包膜组分。包膜含有25%的细胞蛋白，细胞质含有75%。在密度梯度离心后，包膜部分分离成重带（1.23 g/cm 3）和轻带（1.19 g/cm 3）。重带具有高含量的2-酮基-3-脱氧辛酮糖酸（细菌脂多糖的标志物），但没有琥珀酸脱氢酶（细胞质膜活性的标志物），因此代表外膜。较轻的条带表现出高琥珀酸脱氢酶活性，因此包含内（细胞质）膜。用此方法纯化的外膜中细胞质膜的污染率小于5%，而梯度分离的内膜中细胞外膜的污染率高达30%。每个细胞组分的蛋白质组成，其特征在于由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。外膜含有四种主要蛋白质，它们也是整个细胞的主要蛋白质。细胞质膜和可溶性细胞质在凝胶上呈现出更复杂的模式。
Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.