Properties of highly purified leukotriene C4 synthase of guinea pig lung

Abstract

Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized from the microsomes of guinea pig lung and purified 91-fold in four steps to a specific activity of 692 nmol/10 min per mg protein using LTA4-me as substrate. LTC4 synthase of guinea pig lung was separated from microsomal GSH S-transferase by Sepharose CL-4B chromatography and further purified by DEAE-Sephacel chromatography, agarose-butylamine chromatography, and DEAE-3SW fast-protein liquid chromatography. It was also differentiated from the microsomal GSH S-transferase, which utilized 1-chloro-2,4-dinitrobenzene as a substrate, by its heat lability and relative resistance to inhibition by S-hexyl-GSH. The K(m) value of guinea pig lung LTC4 synthase for LTA4 was 3 μM and the V(max) was 108 nmol/3 min per μg; the K(m) values for LTA3 and LTA5 were similar, and the V(max) values were about one-half those obtained with LTA4. The conversion of LYA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective K(i) values of 1.5, 3.3, and 2.8 μM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 μM LTA4-me with 5 mM GSH were 2.1 μM and 0.3 μM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC50 > 40 μM), and LTE4 and LTB4 had no effect on the enzyme, indicating that the mixed type product inhibition observed was specific for sulfidopeptide leukotrienes bearing the GSH moiety.