title: Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits.
abstract: Human leukotriene C4 (LTC4) synthase was purified > 25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholate-solubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (i) anion exchange, (ii) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (iii) gel filtration. The final preparation contained only an 18-kDa polypeptide. The molecular mass of the pure polypeptide was consistent with an 18-kDa polypeptide from THP-1 cell membranes that was specifically photolabeled by an LTC4 photoaffinity probe, 125I-labeled azido-LTC4. On calibrated gel-filtration columns, purified LTC4 synthase activity eluted at a volume corresponding to 39.2 +/- 3.3 kDa (n = 12). The sequence of the N-terminal 35 amino acids was determined and found to be a unique sequence composed predominantly of hydrophobic amino acids and containing a consensus sequence for protein kinase C phosphorylation. We therefore conclude that human LTC4 synthase is a glutathione S-transferase composed of an 18-kDa polypeptide that is enzymatically active as a homodimer and may be phosphoregulated in vivo.
authors.affiliation: Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, PQ, Canada.
authors.forename: D W
authors.initials: DW
authors.lastname: Nicholson
authors.forename: A
authors.initials: A
authors.lastname: Ali
authors.forename: J P
authors.initials: JP
authors.lastname: Vaillancourt
authors.forename: J R
authors.initials: JR
authors.lastname: Calaycay
authors.forename: R A
authors.initials: RA
authors.lastname: Mumford
authors.forename: R J
authors.initials: RJ
authors.lastname: Zamboni
authors.forename: A W
authors.initials: AW
authors.lastname: Ford-Hutchinson
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D066298:In Vitro Techniques; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
chemical_list: D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase