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 mg−1) was 7.6‐fold higher than in U937 cells (0.040 ± 0.017 nmol LTC4 formed · min−1 · mg−1) and comparable to dimethylsulfoxide‐differentiated U937 cells (0.399 ± 0.084 nmol LTC4 formed · min−1 · 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 ≈ LTC4) > (LTD4 ≈ LTE4) > (LTA4 ≈ 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.