Measurement and characterization of membrane-bound and soluble epoxide hydrolase activities in resting mononuclear leukocytes from human blood.
Membrane-bound and soluble epoxide hydrolase activities in the mononuclear cell fraction from human blood have been characterized using cis- and trans-stilbene oxides as substrates, respectively. Because of the low activities in these cells, it was necessary to modify assay procedures developed for rat and mouse liver in the following ways: (a) the substrates were relatively highly labeled (2 Ci/mmol) and carefully purified; (b) the incubation time was extended to 45 to 60 min, during which period the activities were linear; (c) as many as 6 million cells were used for a single assay, which was also within the linear range of the procedure. The membrane-bound epoxide hydrolase characterized in this manner has an apparent Vmax of 7.26 pmol product formed per min per 10(7) cells and an apparent Km of 9.96 microM. The pH optimum was observed to be around 9.8. The dependence of this activity on temperature showed its optimum at 40 degrees. The soluble epoxide hydrolase activity has an apparent Vmax of about 8.26 pmol product formed per min per 10(7) cells, an apparent Km of 1.63 microM, a pH optimum of 6.2 to 6.8, and a temperature optimum at 60 degrees. Using these techniques, these activities have also been determined in other blood components, i.e., lymphocytes, monocytes, granulocytes, erythrocytes, platelets, and plasma. Lymphocytes account for most of the epoxide hydrolase activity towards cis-stilbene oxide, and all of the activity towards trans-stilbene oxide is in the human mononuclear cell fractions. Different substances known to affect rodent epoxide hydrolases were tested for their effects on the human mononuclear blood cell activities. Interestingly, 1,1,1-trichloropropane 2,3-epoxide, a potent inhibitor of liver microsomal epoxide hydrolase in different species including rat, mouse, and human, had little or no effect on the membrane-bound activity measured here. However, cyclohexene oxide inhibits this membrane-bound activity 60%. The soluble epoxide hydrolase is inhibited to 90% of control levels by chalcone epoxide. The membrane-bound and soluble epoxide hydrolase activities determined in 27 subjects varied from 8.2 to 18.5 and from 3.5 to 17.0 pmol product formed per min per 10(7) cells, respectively. The mean coefficient of intraindividual variation, determined with three subjects measured four times each over the course of 18 days, was approximately 10% for both enzyme activities.
D001792:Blood Platelets / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004851:Epoxide Hydrolases / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology; D006098:Granulocytes / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D009000:Monocytes / Q000201:enzymology*
D004851:Epoxide Hydrolases