Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins.
Leukotriene B4 (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 Gbeta 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 (Kd = 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 (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant alpha subunits of Gi1, Gi2, Gi3, and Go 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.[1]References
- Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins. Igarashi, T., Yokomizo, T., Tsutsumi, O., Taketani, Y., Shimizu, T., Izumi, T. Eur. J. Biochem. (1999) [Pubmed]
Annotations and hyperlinks in this abstract are from individual authors of WikiGenes or automatically generated by the WikiGenes Data Mining Engine. The abstract is from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.About WikiGenesOpen Access LicencePrivacy PolicyTerms of Useapsburg
