LM-1484

Kinase experiment:

Equilibrium binding studies are performed at 25°C for 60 min with 0.03-0.5 nM 3H-LTD4 or 40 min with 0.03-0.5 nM 3H-LTC4 and unlabeled homologous or heterologous ligands at the indicated concentrations. A multiligand protocol is followed. Ten micromolar S-decyl-GSH is present only in the case of 3H-LTC4 equilibrium experiments. Time-courses are performed at 25°C with 0.5 nM 3H-LTC4 or 3H-LTD4. Dissociation is induced by adding 1 mM unlabeled leukotriene (homologous dissociation) or 10 μM unlabeled antagonist (heterologous dissociation). In both equilibrium and kinetic studies HLP membranes (0.25 mg per sample), 10 mM HEPES-KOH pH 7.4, 1 mM CaCl2 and 1 mM MgCl2 are added to the incubation mixture to achieve a final volume of 250 mL. All the experiments have been performed under control metabolic conditions. Unbound ligand is separated by rapid vacuum filtration onto glass-fiber GF/C filters soaked in 2.5% polyvinylalchool and the filters are washed twice with 4 mL of HEPES buffer at 4°C. Radioactivity is measured in a liquid scintillation counter.

References:

[1]. Ravasi S, et al. Pharmacological differences among CysLT(1) receptor antagonists with respect to LTC(4) and LTD(4) in human lung parenchyma. Biochem Pharmacol. 2002 Apr 15;63(8):1537-46.