Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells.
Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4 mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at > or = 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC2-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83 mM and 19.6 microM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2-4 mumol LTC4 formed .min-1 x mg-1.
Nicholson
D W
DW
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Klemba
M W
MW
Rasper
D M
DM
Metters
K M
KM
Zamboni
R J
RJ
Ford-Hutchinson
A W
AW
D002454:Cell Differentiation / Q000187:drug effects; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D004121:Dimethyl Sulfoxide