Purification of human leukotriene C4 synthase.
Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 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-hexyl-glutathione-agarose column that was eluted by the use of 7.5 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 approximately 75 mg of S-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 of the 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 approximately 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.
Penrose
J F
JF
Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA.
Gagnon
L
L
Goppelt-Struebe
M
M
Myers
P
P
Lam
B K
BK
Jack
R M
RM
Austen
K F
KF
Soberman
R J
RJ
D002846:Chromatography, Affinity; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification*; D006801:Humans; D066298:In Vitro Techniques; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D011339:Probenecid / Q000494:pharmacology; D013189:SRS-A / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D013189:SRS-A; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione; D011339:Probenecid