Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung.
Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.
Manganaro
F
F
Bio-Méga, Inc., Laval, Québec, Canada.
Gaudette
Y
Y
Pombo-Gentile
A
A
Singh
K
K
Rakhit
S
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000302:isolation & purification*; D066298:In Vitro Techniques; D007700:Kinetics; D017572:Leukotriene A4; D007975:Leukotriene B4 / Q000096:biosynthesis; D015289:Leukotrienes / Q000096:biosynthesis / Q000378:metabolism*; D008168:Lung / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D017572:Leukotriene A4; D015289:Leukotrienes; D007975:Leukotriene B4; D012710:Serum Albumin, Bovine; D004851:Epoxide Hydrolases