Properties of highly purified leukotriene C4 synthase of guinea pig lung

T. Yoshimoto, R. J. Soberman, B. Spur, K. F. Austen

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78 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)866-871
Number of pages6
JournalJournal of Clinical Investigation
Volume81
Issue number3
DOIs
StatePublished - 1988
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General Medicine

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