title: Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells.
abstract: Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
authors.forename: M
authors.initials: M
authors.lastname: Abe
authors.forename: T E
authors.initials: TE
authors.lastname: Hugli
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001202:Ascitic Fluid / Q000166:cytology / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D005122:Exudates and Transudates / Q000166:cytology / Q000201:enzymology*; D005982:Glutathione Transferase / Q000378:metabolism*; D017572:Leukotriene A4; D051379:Mice; D013189:SRS-A / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013997:Time Factors
chemical_list: D001095:Arachidonic Acids; D004137:Dinitrochlorobenzene; D017572:Leukotriene A4; D013189:SRS-A; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase