Castle J D, Jamieson J D, Palade G E
J Cell Biol. 1975 Jan;64(1):182-210. doi: 10.1083/jcb.64.1.182.
A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.
通过一种特别有效的方法从兔腮腺中分离出了一部分分泌颗粒，该方法能有效减少颗粒与其他细胞颗粒聚集和/或共沉降所导致的污染。该部分颗粒平均占总组织淀粉酶活性的15%，经电子显微镜判断其均一，通过标记酶法和化学分析法判断，其他细胞器对其污染程度极低。通过将颗粒逐渐暴露于含有0.5 mM EDTA的低渗NaHCO₃中实现颗粒裂解。颗粒裂解物经离心分离得到的内容物和膜主要通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳进行表征，结果表明内容物由有限数量分子量级别的多肽组成，其中三条带（分子量约为58,000、33,000和12,000）可被视为主要成分。膜亚组分的凝胶图谱特征是有20 - 30条考马斯亮蓝染色带，其中一种分子量为40,000的单一物种是明显的主要多肽。进行了两种采用凝胶电泳分析的实验，用于鉴定和评估吸附在纯化颗粒膜上的残留分泌蛋白的程度：（a）将放射性分泌颗粒提取物与非放射性标记的颗粒膜混合后检查染色和放射性图谱；（b）将分泌颗粒提取物和颗粒膜的凝胶图谱与未裂解的分泌颗粒以及体外从小叶中排出或通过腮腺导管插管收集的分泌蛋白的凝胶图谱进行比较，后两个样品被视为生理分泌标准。结果表明，尽管对膜部分进行典型分泌酶活性（淀粉酶）测定表明膜和内容物已相当彻底地分离，但膜仍被残留的、难以提取的分泌蛋白大量污染。因此，通过凝胶电泳对膜亚组分中的残留内容物种类进行详细检查，表明该技术作为一种准确检测细胞组分或细胞器亚组分中作为污染物出现的特定多肽组的手段具有普遍的通用性和敏感性。
