Purification of human lung leukotriene C4 synthase and preparation of a polyclonal antibody.

Abstract

Leukotriene (LT) C4 synthase is an integral membrane protein that catalyzes the conjugation of LTA4 to reduced glutathione to form LTC4. LTC4 synthase has been cloned and characterized from transformed cell lines, but the protein has not been defined from a tissue source. LTC4 synthase was purified to homogeneity from human lung tissue, utilizing S-hexyl glutathione chromatography followed by LTC4 affinity chromatography. A greater than 100,000-fold purification with a yield of 8 to 25% (n = 4) was achieved. The purified LTC4 synthase migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as an 18-kD protein, and its 19 N-terminal amino acid sequence is identical to that of purified LTC4 synthase from KG-1 myeloid cells or from expression cloning of a KG-1 library in COS cells. Using a rabbit polyclonal IgG raised against purified LTC4 synthase, SDS-PAGE immunoblotting of LTC4 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-LTC4 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.

Role of Group V Phospholipase A2 in Zymosan-induced Eicosanoid Generation and Vascular Permeability Revealed by Targeted Gene Disruption

“…2). The perinuclear region is a prominent location for enzymes of eicosanoid biosynthesis, either constitutively (48,49) or after translocation in response to calcium flux (4,50,51). Thus, group V sPLA 2 may act intracellularly, without a requirement for prior secretion, to amplify the effects of translocated cPLA 2 -␣.…”

Activation of human eosinophils through leukocyte immunoglobulin-like receptor 7

“…Cytosolic phospholipase A 2 and 5-LO exist in the cytosol and translocate to the perinuclear membrane in response to calcium flux (29,30). 5-LO-activating protein and LTC 4 synthase are present at the perinuclear membrane in both resting and stimulated cells (31,32). Subsequent work implicated lipid bodies as a site of LT biosynthesis (26).…”

Asthma and leukotrienes: antileukotrienes as novel anti‐asthmatic drugs

“…Immunohistochemical analysis of human lung showed that LTC 4 synthase was localized to perinuclear membrane in alveolar macrophages [72]. Recently, it was reported that 5-lipoxygenase and LTC 4 synthase were assembled in lipid bodies in platelet activating factor (PAF) stimulated eosinophils [73], demonstrating cell variation in the localization of the enzymes involved in leukotriene synthesis.…”