Purification of human leukotriene C4 synthase.

Abstract

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation ofLTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexylglutathione-agarose column that was eluted by the use of7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from -75 mg ofS-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder ofthe eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only -1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.

Characterization of the ATP‐dependent leukotriene C4 export carrier in mastocytoma cells

“…Incubation of LTA, with mastocytoma membrane vesicles in the presence of glutathione resulted in the formation of LTC, (unpublished results), indicating the presence of LTC, synthase as another potential LTC,-binding protein in our membrane preparation. LTC, synthase was found to be a unique membrane-bound enzyme distinct from other cytosolic and microsomal glutathione S-transferases [36,37,[46][47][48]. Recent studies in dimethylsulfoxide-differentiated U 937 cells indicated that human LTC, synthase is composed of an 18-kDa protein that is enzymically active as a homodimer and contains a phosphorylation site [36,37,491.…”

Leukotrienes, Sphingolipids, and Leukocyte Trafficking

“…LTC 4 is formed by conjugation of LTA 4 with the tripeptide glutathione through the catalysis of LTC 4 synthase. Stimulated by divalent cations and phosphatidylcholine, LTC 4 synthase is an 18-kDa protein with a wide tissue distribution (31,32). After LTC 4 synthesis, the multidrug transporter ATP-binding cassette (Abc)c1 (formerly known as MRP-1) actively transports LTC 4 out of the cell, where LTD 4 and LTE 4 are formed through the elimination of glutamine and glycine, respectively, by ␥-glutamyl transpeptidase and dipeptidase (33)(34)(35).…”

The membrane organization of leukotriene synthesis

“…FLAP may serve as an arachidonic acidbinding protein, allowing arachidonic acid to be presented to 5-LO for conversion to both 5-HPETE and LTA 4 (14)(15)(16)(17)(18). To form LTC 4 , LTA 4 is conjugated with reduced glutathione by LTC 4 synthase, a 17-kDa integral membrane protein that shares a high identity with FLAP (19)(20)(21)(22)(23). Two untested models for the membrane organization of LTC 4 synthesis can be proposed.…”