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

点击翻译按钮获取中文摘要

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.