Photoaffinity labelling of human leukotriene C4 synthase in THP-1 cell membranes.
Human leukotriene C4 synthase specific activity in the human monocytic leukemia cell line THP-1 (0.302 +/- 0.062 nmol LTC4 formed.min-1 x mg-1) was 7.6-fold higher than in U937 cells (0.040 +/- 0.017 nmol LTC4 formed.min-1 x mg-1) and comparable to dimethylsulfoxide-differentiated U937 cells (0.399 +/- 0.084 nmol LTC4 formed.min-1 x mg-1). Using the photoaffinity probe, azido[125I]-LTC4, a single polypeptide with a molecular mass of 18 kDa was specifically labelled in THP-1 microsomal membranes. The rank order of potencies for competition of azido[125I]-LTC4 photolabelling of the 18 kDa protein by glutathione, leukotrienes and their analogs was found to be LTC2 > (azido[127I]-LTC4 approximately LTC4) > (LTD4 approximately LTE4) > (LTA4 approximately LTB4) > S-hexyl glutathione > glutathione, corresponded with the rank order of potencies for inhibition of LTC4 synthase activity but not inhibition of microsomal glutathione S-transferase activity. The 18 kDa protein specifically labelled by azido[125I]-LTC4 had high specificity for LTC4 and closely related leukotrienes and was separable from microsomal glutathione S-transferase. We conclude that azido[125I]-LTC4 specifically photolabels LTC4 synthase which is an 18 kDa polypeptide or contains an 18 kDa subunit.
Ali
A
A
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
Zamboni
R J
RJ
Ford-Hutchinson
A W
AW
Nicholson
D W
DW
D000345:Affinity Labels; D001667:Binding, Competitive; D002460:Cell Line; D005982:Glutathione Transferase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009000:Monocytes / Q000201:enzymology; D010777:Photochemistry; D012995:Solubility; D013656:Taurocholic Acid
D000345:Affinity Labels; D013656:Taurocholic Acid; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase