Isolation of Macrophage Mutant Defective in LPS Receptor and Purification of the Receptor

从巨噬细胞样小鼠细胞系J774.1中分离出对LPS无应答的LPS抗性突变体。与亲本J774.1细胞不同，这些突变体甚至在添加LPS的培养基中以及在正常生长培养基中生长而没有任何形态学变化。[ ^<125>I] LPS与细胞单层结合的测定显示，这些LPS抗性突变体之一（LR-9）在LPS结合活性方面存在显著缺陷。Scatchard图显示，LR-9细胞缺乏J774.1细胞的高亲和力结合位点。LPS可促进J774.1细胞的O_2 ~-生成和花生四烯酸的释放，但对LR-9细胞无明显影响。其他刺激物如酵母多糖和TPA则能诱导LR-9细胞和J774.1细胞释放花生四烯酸，LPS光交联法可在J774.1细胞膜组分中鉴定出65 kDa和55 kDa的LPS结合蛋白。这两条带在LR-9细胞中均未检测到，并在与未标记的LPS或脂质X竞争时消失。这些结果表明，LPS结合蛋白中的一个或两个可能与LPS的特异性膜受体有关。纯化的蛋白质由65 kDa和55 kDa的蛋白质组成。

LPS-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of [ ^<125>I] LPS-binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1.LPS enhanced O_2^- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and TPA, however, induced the release of arachidonic acid in LR-9 cells as well a in J774.1 cells.LPS-photo-cross-linked assay allowed the identification of 65 kDa and 55 kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabelled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.LPS receptor was solubilized with SDS and then purified by gel filtrarion column and LPS-affinity column. The purified protein was consisted of 65 kDa and 55 kDa proteins.