ISOLATION AND IDENTIFICATION OF THE LPS BINDING PROTEINS ON HUMAN MONOCYTES

通过使用两种不同的方法，我们已经分离了几种蛋白质 它们可能是人单核细胞上的内毒素结合蛋白。一种方法 将内毒素加入35-S-蛋氨酸标记的单核细胞中，裂解细胞 然后，使用抗内毒素抗体，免疫沉淀内毒素 与任何结合的单核细胞蛋白。我们发现了两种蛋白质，一种 超过200千米和一个大约100千米的场地。越大 经Western印迹分析，该蛋白为CD45。 110kd的蛋白质尚未被鉴定，但我们已经发现 经干扰素-γ处理后，其表达增强。第二 使用的方法包括将脂多糖共价连接到CNBr活化的琼脂糖凝胶 珠子。然后将珠子加入标记为35-S-蛋氨酸的裂解产物中 单核细胞，洗涤几次以去除不结合的蛋白质 内毒素。使用这项技术，我们发现了另外两种蛋白质 约为70和90kd。目前正在进行的工作是 鉴定所有这些蛋白并验证它们在内毒素诱导中的作用 人单核细胞的激活。

By utilizing two different approaches, we have isolated several proteins which may serve as LPS binding proteins on human monocytes. One method used was to add LPS to 35-S-methionine labeled monocytes, lyse the cells and then, using an anti-LPS antibody, immunoprecipitate the LPS along with any bound monocyte proteins. We have found two proteins, one greater than 200 kd and one approximately a 100 kd in site. The greater than 200 kd protein has been identified by Western blot analysis as CD45. The 110 kd protein has not yet been identified, but we have found that its expression is enhanced by pretreatment with IFN-gamma. The second approach used involved covalently linking LPS to CNBr-activated sepharose beads. The beads are then added to a lysate of 35-S-methionine labeled monocytes and washed several times to remove proteins which do not bind the LPS. Using this technique, we have found two additional proteins which are approximately 70 and 90 kd. Work is currently in progress to identify all these proteins and to verify their role in the LPS induced activation of human monocytes.