Mellman I S, Steinman R M, Unkeless J C, Cohn Z A
J Cell Biol. 1980 Sep;86(3):712-22. doi: 10.1083/jcb.86.3.712.
We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.
我们描述了一种基于乳过氧化物酶（LPO）和葡萄糖氧化酶（GO）同时内吞作用的对胞饮小泡（PVs）进行特异性放射性碘化的方法。初步实验表明，巨噬细胞系J774通过液相胞饮作用内化LPO，且未检测到其与质膜（PM）的结合。内化作用随酶浓度和暴露时间呈线性变化，依赖于温度，在4℃时无法检测到。采用电子显微镜细胞化学方法，在37℃脉冲3至5分钟后，LPO活性局限于PVs。这些结果构成了碘化PVs腔表面方法的基础：在37℃下同时暴露于LPO和GO 5分钟，随后洗涤并在4℃下进行碘化（加入125I和葡萄糖）。发生了酶依赖性的碘化物掺入PV膜和内容物的多肽中。几条证据表明，与PM相反，PV有选择性标记。如果低温抑制LPO和GO的胞饮摄取，则不会发生碘化。电子显微镜放射自显影显示颗粒位于细胞质中，而表面标记时质膜有明显的结合。LPO仅在PV标记后被碘化，并存在于显示潜伏性的细胞器内。PV碘化后，几种标记的膜抗原中超过75%只有在细胞裂解后才能被单克隆抗体免疫沉淀。相比之下，表面标记后完整细胞上的所有标记抗原均可被抗体识别。通过SDS聚丙烯酰胺凝胶电泳和使用一组抗J774单克隆抗体的定量免疫沉淀法比较了PM和PV膜的多肽组成。在PV和PM碘化细胞的NP-40裂解物中，碘化蛋白的电泳图谱（15至20条带）惊人地相似。此外，通过免疫沉淀检测的八种膜抗原，包括抗胰蛋白酶免疫球蛋白（Fc）受体和H-2Dd组织相容性抗原，在两种标记程序中被碘化的相对程度相同。我们得出结论，PV膜由PM多肽成分的代表性样本形成。
