Purification of human lung leukotriene C4 synthase and preparation of a polyclonal antibody.
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.
Penrose
J F
JF
Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Spector
J
J
Lam
B K
BK
Friend
D S
DS
Xu
K
K
Jack
R M
RM
Austen
K F
KF
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D008168:Lung / Q000201:enzymology*; D016676:Macrophages, Alveolar / Q000201:enzymology; D008969:Molecular Sequence Data
D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase