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 CI-4B chromatography and further purified by DEAESephacel 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 Shexyl-GSH. The K. value of guinea pig lung LTC4 synthase for LTA. was 3 ,uM and the V., was 108 nmol/3 min per gg, the K, values for LTA3 and LTA5 were similar, and the V., values were about one-half those obtained with LTA4. The conversion of LTA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective K, values of 1.5, 3.3, and 2.8 MM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 ,uM LTA4-me with 5 mM GSH were 2.1 ,uM and 0.3 MuM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC5o > 40 MuM), 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.

Characterization of the ATP‐dependent leukotriene C4 export carrier in mastocytoma cells

“…Incubation of LTA, with mastocytoma membrane vesicles in the presence of glutathione resulted in the formation of LTC, (unpublished results), indicating the presence of LTC, synthase as another potential LTC,-binding protein in our membrane preparation. LTC, synthase was found to be a unique membrane-bound enzyme distinct from other cytosolic and microsomal glutathione S-transferases [36,37,[46][47][48]. Recent studies in dimethylsulfoxide-differentiated U 937 cells indicated that human LTC, synthase is composed of an 18-kDa protein that is enzymically active as a homodimer and contains a phosphorylation site [36,37,491.…”

GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D 4

“…inflammation ͉ knockout mice ͉ lipid mediator ͉ macrophage T he cysteinyl leukotrienes (cys-LTs), leukotriene (LT) C 4 , LTD 4 , and LTE 4 , are proinflammatory mediators generated by the 5-lipoxygenase (5-LO) pathway after activation of particular bone marrow-derived cells to release arachidonic acid from the phospholipids of the outer nuclear membrane. In the presence of the 5-LO-activating protein (1, 2), 5-LO converts arachidonic acid to LTA 4 (3), which can be conjugated to reduced glutathione to form LTC 4 by an integral trimeric nuclear membrane enzyme, LTC 4 synthase (4 -6).…”

“…In the presence of the 5-LO-activating protein (1, 2), 5-LO converts arachidonic acid to LTA 4 (3), which can be conjugated to reduced glutathione to form LTC 4 by an integral trimeric nuclear membrane enzyme, LTC 4 synthase (4 -6). After energydependent export of LTC 4 , glutamic acid and glycine are sequentially cleaved by ␥-glutamyl transpeptidase (7) or ␥-glutamyl leukotrienase (8) and dipeptidases (9,10) to form LTD 4 and LTE 4 , respectively.…”

“…In the presence of the 5-LO-activating protein (1, 2), 5-LO converts arachidonic acid to LTA 4 (3), which can be conjugated to reduced glutathione to form LTC 4 by an integral trimeric nuclear membrane enzyme, LTC 4 synthase (4 -6). After energydependent export of LTC 4 , glutamic acid and glycine are sequentially cleaved by ␥-glutamyl transpeptidase (7) or ␥-glutamyl leukotrienase (8) and dipeptidases (9,10) to form LTD 4 and LTE 4 , respectively. The cys-LTs are implicated in human bronchial asthma by their pharmacologic actions to constrict airway and vascular smooth muscle (11)(12)(13) and by the clinical efficacy of agents that block 5-LO or the type 1 receptor for the cys-LTs (CysLT 1 R) (14,15).…”

Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inflammation and fibrosis

“…An integral perinuclear membrane protein, LTC 4 synthase (LTC 4 S), conjugates reduced glutathione to LTA 4 to form LTC 4 (13,14). After carrier-mediated export (15), LTC 4 is converted to additional receptor-active metabolites, LTD 4 and LTE 4 , by the sequential cleavage from the tripeptide adduct of glutamate (16,17) and glycine.…”