Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells
Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells
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DOI：
10.1006/abio.1995.0010
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发表时间：
1995-12-10
影响因子：
2.9
通讯作者：
Staunton, D
中科院分区：
文献类型：
--
作者：
Luk, JM;Kumar, A;Staunton, D
内毒素或脂多糖(LPS)是革兰氏阴性菌的主要细胞表面成分，释放到血液中后可与不同类型的细胞结合，在感染性休克综合征的发病机制中起着核心作用。我们建立了一种从明尼苏达沙门氏菌R595和大肠杆菌J5细菌中纯化的脂多糖分子的生物素化标记方法，生物素基团通过化学氧化脂多糖碳水化合物部分(内核区)，然后用生物素-LC-酰肼(生物素-己酰肼)还原，或通过与生物素-LC-AsA[1-(4-azidosalicylamido-)-6(biotinamido)-hexane]，的光活化交联法与细菌脂多糖偶联，生物素-LC-AsA DNA随机连接到脂多糖的碳水化合物和脂肪酸(脂A)基团。两种标记产物都保持了生物活性(或内毒素)，通过凝集变形细胞裂解物来确定其与亲和素/链霉亲和素的结合能力，该亲和素/链霉亲和素可与酶和荧光探针偶联。将生物素化的脂多糖用于酶免疫分析、Western印迹和流式细胞术，并分析了内毒素配体与其对位受体(S)在全细胞上的结合情况。生物素化的脂多糖探针可应用于受体生物化学、免疫组织化学和分子细胞生物学中的多种技术。(C)1995年学术出版社。
Endotoxin or lipopolysaccharide (LPS), a major cell surface component of gram-negative bacteria, which could bind to different cell types when released into the bloodstream, plays a central role in the pathogenesis of septic shock syndromes. We have established a biotinylation procedure for labeling purified LPS molecules from Salmonella minnesota R595 and Escherichia coli J5 bacteria, The biotin group was conjugated to the bacterial LPS either by chemical oxidation of the LPS carbohydrate moiety (inner core region), followed by reduction with biotin-LC-hydrazide (biotinamido hexanoyl hydrazide), or by photoactivatable cross-linking with biotin-LC-ASA [1-(4-azidosalicylamido-)-6(biotinamido)-hexane], which was randomly attached to the carbohydrate and fatty acid (lipid A) groups of the LPS. Both labeled products retained biological activity (or endotoxicity) as evidenced by coagulation of the Limulus amoebocyte lysate, To determine its ability to bind avidin/streptavidin which in turn could be conjugated with enzymatic and fluorescent probes, the biotinylated LPS was used in enzyme immunoassay, Western blot, and flow cytometry, These assays were also used to analyze the binding of LPS ligand to its counterreceptor(s) on whole cell. surface, membrane fragments, and in detergent lysates from human endothelial and monocytic cells, The described biotinylated LPS probes can be applied in a wide variety of techniques in receptor biochemistry, immunohistochemistry, and molecular cell biology. (C) 1995 Academic Press, Inc.