Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells.
Leukotriene C4 (LTC4) synthase was highly expressed in the human U937 monoblast leukemia cell line when differentiated into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide. The specific activity of LTC4 synthase in differentiated cells (399.0 +/- 84.1 pmol of LTC4 formed.min-1.mg-1) was markedly higher (10-fold; p less than 0.001) than in undifferentiated U937 cells (39.9 +/- 16.7 pmol of LTC4 formed.min-1.mg-1) or freshly isolated blood monocytes (21.5 +/- 4.8 pmol of LTC4 formed.min-1.mg-1). The increase in LTC4 synthase activity following dimethyl sulfoxide-induced differentiation was substantially higher than the increase observed for other proteins involved in leukotriene biosynthesis. LTC4 synthase activity was unaffected in U937 cells differentiated by growth in the presence of phorbol 12-myristate 13-acetate. The HL-60 myeloblast leukemia cell line expressed higher LTC4 synthase levels when differentiated into either neutrophil-like or macrophage-like cells by growth in the presence of dimethyl sulfoxide or phorbol 12-myristate 13-acetate (respectively), but reached a specific activity comparable only to undifferentiated U937 cells. Human LTC4 synthase was found to be a unique membrane-bound enzymatic activity completely distinct from alpha, mu, pi, theta, and microsomal glutathione S-transferases, as determined by differential detergent solubilization, chromatographic separation, substrate specificity, and Western blot analysis. An 18-kDa polypeptide was specifically labeled in membranes from dimethyl sulfoxide-differentiated U937 cells using azido 125I-LTC4, a photoaffinity probe based on the product of the LTC4 synthase-catalyzed reaction. Photolabeling of the 18-kDa polypeptide was specifically competed for by LTC4 (greater than 50% at 0.1 microM) but not by 100,000-fold higher concentrations of reduced glutathione (10 mM). Elevation of both the level of the specifically photolabeled 18-kDa polypeptide and of LTC4 synthase specific activity occurred concomitantly with dimethyl sulfoxide differentiation of U937 cells. We conclude that differentiation of U937 cells into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide results in high levels of expression of LTC4 synthase activity. Human LTC4 synthase is a unique enzyme with a high degree of specificity for LTA4 and may therefore be dedicated exclusively to the formation of LTC4 in vivo. An 18-kDa membrane polypeptide, specifically labeled by a photoaffinity derivative of LTC4, is a candidate for being either LTC4 synthase or a subunit thereof.
Nicholson
D W
DW
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Ali
A
A
Klemba
M W
MW
Munday
N A
NA
Zamboni
R J
RJ
Ford-Hutchinson
A W
AW
D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division; D002458:Cell Fractionation; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism*; D006098:Granulocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D013189:SRS-A / Q000096:biosynthesis / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000201:enzymology; D014407:Tumor Cells, Cultured
D013189:SRS-A; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D004121:Dimethyl Sulfoxide