Characterization of the soluble leukotriene B4 receptor from sheep lung membranes.
Leukotriene B4 (LTB4) is an arachidonate metabolite which elicits a variety of pro-inflammatory responses by activation of a guanine-nucleotide-binding protein-coupled membrane receptor. As a prelude to receptor isolation and purification, we have established assay methods for LTB4 receptor solubilization and characterization from sheep lung membranes. [3H]LTB4 binding to the soluble receptor was saturable, specific, protein-concentration- and time-dependent and reversible. Binding of [3H]LTB4 was enhanced by divalent cations and inhibited by sodium ions in a manner analogous to its binding to the human leukocyte membrane receptor. Saturation binding yielded a dissociation constant (Kd) of 0.50 +/- 0.05 nM and a receptor density (Bmax) of 330 +/- 90 fmol/mg of protein for [3H]LTB4 binding to detergent-solubilized receptor. In competition experiments, the rank order of binding affinity was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than U-75302. Gel-filtration chromatography showed that the LTB4 receptor protein in the detergent micellar state has a molecular mass in the range 800-1000 kDa. These results demonstrate that the physiologically and pharmacologically important LTB4 receptor may be readily solubilized from sheep lung membranes without alteration in binding specificity and characteristics, suggesting that sheep lung membranes represent a rich source with which to pursue receptor isolation and purification.
Votta
B
B
Department of Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939.
Keefer
J
J
Mong
S
S
D000818:Animals; D001667:Binding, Competitive; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004492:Edetic Acid / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D007975:Leukotriene B4 / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000378:metabolism*; D018102:Receptors, Leukotriene B4; D012756:Sheep; D012995:Solubility; D013873:Thionucleotides / Q000494:pharmacology
D002412:Cations; D003902:Detergents; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D013873:Thionucleotides; D007975:Leukotriene B4; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004492:Edetic Acid