PURIFICATION OF HUMAN LUNG LEUKOTRIENE C-4 SYNTHASE AND PREPARATION OF A POLYCLONAL ANTIBODY

Leukotriene (LT) C-4 synthase is an integral membrane protein that catalyzes the conjugation of LTA(4) to reduced glutathione to form LTC(4). LTC(4) synthase has been cloned and characterized from transformed cell lines, but the protein has not been defined from a tissue source. LTC(4) synthase was purified to homogeneity from human lung tissue, utilizing S-hexyl glutathione chromatography followed by LTC(4) affinity chromatography. A greater than 100,000-fold purification with a yield of 8 to 25% (n = 4) was achieved. The purified LTC(4) synthase migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PACE) as an 18-kD protein, and its 19 N-terminal amino acid sequence is identical to that of purified LTC(4) synthase from KC-1 myeloid cells or from expression cloning of a KG-1 library in COS cells. Using a rabbit polyclonal IgC raised against purified LTC(4) synthase, SDS-PAGE immunoblotting of LTC(4) synthase from human lung tissue, eosinophils, KG-1 cells, and platelets showed an 18-kD protein. Immunofluorescence staining of alveolar macrophages in human lung sections with the anti-LTC(4) synthase IgG revealed LTC4 synthase to be largely perinuclear in distribution. Thus, LTC4 synthase, the biosynthetic enzyme responsible for the formation of cysteinyl LTs, is present in lung tissue in a form apparently identical to that of hematopoietic cells.