Lipid A binding sites in membranes of macrophage tumor cells.
Lipid A binding sites in membranes of macrophage tumor cells.
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DOI：
10.1016/s0021-9258(18)68109-8
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发表时间：
1988-10
期刊：
影响因子：
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通讯作者：
R. Hampton;D. Golenbock;C. H. Raetz
中科院分区：
文献类型：
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作者：
R. Hampton;D. Golenbock;C. H. Raetz
脂多糖影响多种真核细胞和哺乳动物生物体。这些作用与革兰氏阴性败血症的发病机制有关。脂多糖的许多作用被认为是由其活性部分脂质 A 引起的。我们的实验室先前已鉴定出一种生物活性脂质 A 前体，称为脂质 IVA（Raetz, C. R. H.、Purcell, S.、Meyer, M. V.、Qureshi, N. 和 Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888），它可以是用高比活性的 32P 标记并纯化。在这项工作中，我们使用标记探针 4'-32P-脂质 IVA 开发了一种新的测定方法，用于脂质 IVA 与全细胞的特异性结合。我们还证明了其在固定化细胞蛋白的配体印迹测定中的用途。使用全细胞测定，我们表明 4'-32P-脂质 IVA 特异性结合 RAW 264.7 巨噬细胞样培养细胞。结合是饱和的，会被过量的未标记脂质 IVA 抑制，并且对蛋白酶 K 敏感。它显示出细胞和药理学特异性。使用配体印迹测定，我们表明几种 RAW 264.7 细胞蛋白可以结合 4'-32P-脂质 IVA。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断，两种主要结合蛋白的 Mr 值分别为 31 和 95 kDa。分级分离研究表明，31-kDa 蛋白富集在核部分，可能是组蛋白，而 95-kDa 蛋白富集在膜部分。我们开发的结合测定应该可以让我们更清楚地了解脂质 A/动物细胞的相互作用。
Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4′-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4′-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4′-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.