1999年1月
Characterization of the leukotriene B-4 receptor in porcine leukocytes - Separation and reconstitution with heterotrimeric GTP-binding proteins
EUROPEAN JOURNAL OF BIOCHEMISTRY
- ,
- ,
- ,
- ,
- ,
- 巻
- 259
- 号
- 1-2
- 開始ページ
- 419
- 終了ページ
- 425
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1046/j.1432-1327.1999.00054.x
- 出版者・発行元
- BLACKWELL SCIENCE LTD
Leukotriene B-4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide-binding regulatory protein (G-protein)coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G-proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G-proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G-proteins, in combination with the fraction which contained endogenous phospholipids and G(beta gamma). Kinetic studies of LTB4 were performed to determine the association with G-proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G-proteins showed a lower affinity for LTB4 (K-d = 500 nM), but reconstitution of the BLT fraction with a G-protein-rich fraction (flow-through of an RCA column) increased the affinity for LTB4 10-fold (K-d = 50 nM). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric G(i2) purified from bovine brain or recombinant or subunits of G(i1), G(i2), G(i3), and G(o) expressed in Spodoptera frugiperda-9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin-sensitive G-proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G-protein-coupled receptors.
- リンク情報
- ID情報
-
- DOI : 10.1046/j.1432-1327.1999.00054.x
- ISSN : 0014-2956
- Web of Science ID : WOS:000077944300054