Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase.
A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D005982:Glutathione Transferase / Q000032:analysis*; D005779:Immunodiffusion; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008433:Mathematics; D008970:Molecular Weight; D010777:Photochemistry; D051381:Rats; D013189:SRS-A / Q000378:metabolism*; D013379:Substrate Specificity; D014018:Tissue Distribution
D000345:Affinity Labels; D013189:SRS-A; D005982:Glutathione Transferase