Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells.

AUTOR(ES)

Yoshimoto, T

RESUMO

When leukotriene (LT) A4 was incubated with subcellular fractions of sonicated rat basophilic leukemia (RBL) cells in the presence of glutathione, the enzyme producing LTC4, designated LTC4 synthetase, was found in the 105,000 X g pellet (microsomes) with a 3-fold enrichment in specific activity over that of the sonicate. The identification of the reaction product as LTC4 was confirmed by its identical retention time on reverse-phase HPLC to that of synthetic LTC4, the incorporation of [3H]glutathione into the product, its reactivity in a radioimmunoassay, and its UV absorption spectrum. In contrast, glutathione S-transferase activity, measured spectrophotometrically with 1-chloro-2,4-dinitrobenzene, was detected predominantly in the 105,000 X g supernatant (89%) and also in the microsomes (7%). The microsomal glutathione S-transferase and LTC4 synthetase were solubilized with 0.4% Triton X-102 and separated by DEAE-Sephacel chromatography; the former appeared in the effluent and the latter in the eluate after the addition of 0.16 M NaCl to the equilibration buffer. Solubilized, microsomal glutathione S-transferase was inhibited by S-hexylglutathione with an IC50 of 36 microM and was stable at 40 degrees C for 5 min, whereas LTC4 synthetase was only slightly inhibited (IC50, 2.3 mM) by S-hexylglutathione and retained no activity after incubation at 40 degrees C for 5 min. The partially purified LTC4 synthetase showed a specific activity of 1.34 +/- 0.51 nmol of LTC4 per 10 min per mg of protein (mean +/- SD, n = 9), representing a 10-fold purification from the sonicate and catalyzed the dose- and time-dependent production of LTC4 from LTA4 and glutathione. The apparent Km values for LTA4 and glutathione were estimated by Lineweaver-Burk plots to be 5-10 microM and 3-6 mM, respectively. These results indicate that the conjugation of LTA4 with glutathione to form LTC4 is catalyzed by a unique microsomal enzyme.

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