Characterization of specific binding sites for cysteinyl leukotrienes in sheep lung.

Specific binding sites for [3H]leukotriene (LT)D4 and [3H]LTC4 have been identified in sheep lung parenchymal membranes. [3H] LTD4 specific binding was of high affinity (KD = 0.56 nM), saturable (Bmax = 43 fmol/mg of protein), stimulated by divalent cations and inhibited by nonhydrolyzable GTP analogs. LTs and LTD4-receptor antagonists competed for [3H]LTD4 specific binding with the rank order of potency predicted for the LTD4 receptor: LTD4 > ONO-1078 > ICI 204,219 > MK-571 > LTE4 > LTC4 > BAY u9773 >> LTB4. In contrast, [3H]LTC4 specific binding was of lower affinity (KD = 27 nM), abundant (Bmax = 87 pmol/mg of protein) and although stimulated by divalent cations was unaffected by GTP analogs. LTs and LTC4 analogs competed for [3H]LTC4 specific binding with the following rank order of potency: LTC2 > LTC3 > LTC4 > LTC5 >> N-methyl-LTC4 >> LTD4 approximately LTB4 approximately LTB4. [3H]LTD4 specific binding to sheep lung membranes has, therefore, the characteristics of being to a G-protein-coupled LTD4 receptor, whereas the profile of [3H]LTC4 specific binding strongly suggests that these sites are not LT-receptor related. Photolabeling of sheep lung membranes using [125I]azido-LTC4, a photoactivable LTC4 analog, resulted in the selective photolabeling of two polypeptides migrating at 30 kDa and 19 kDa. The selective photolabeling of the 19 kDa polypeptide could be modulated in an identical manner to [3H]LTC4 specific binding. This protein is, therefore, a candidate for being the principal [3H]LTC4 specific site in sheep lung membranes and has a comparable molecular mass to microsomal glutathione S-transferase, recently shown to be the predominant LTC4 binding protein in cellular membranes.