Abstract
Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.
Highlights
Human erythrocytescontainedasolublecytosolic hydration of LTA, produces LTB4 [6, 7], a chemokinetic, epoxide hydrolase for stereospecific enzymatic hydram- yotropic, dihydrodiol ((5S,12R)-5,12-dihydroxy-6,14-cistion of leukotriene A, into leukotrieneB4.The enzyme 8,lO-trans-eicosatetraenoicacid) implicated in inflammatory was purified 1 lOO-foId, to apparent electrophoretic and respiratory disorders [8,9,10].In the absence of epoxide homogeneity, by conventional DEAE-SephaceFfrac- hydrolase, spontaneous, nonenzymatic hydration of the untionationfollowedbyhighperformanceanion ex- stable substrate (LTA4)occurs
In contrast to other cells, leukocytes [6, 7, 14,15,16,17], erythrocytes have seldom been attributed any capacity for eicosanoid biosynthesis until recently (18, 19F)u.rthermore, epoxidesincluding (+)-14,15-oxido-5,8,ll-eicosatri- erythrocytes have not been recognized as a source of extraenoicacidand (f)-l1,12-oxido-5,8,14-eicosatrienoic hepatic epoxide hydrolase activity for hydration of xenobiotic acid, but not others, including 5,6-oxido-8,11,14-ei- oxiranes [2]
Cellular Localization-Epoxide hydrolase activity was detected in thecytosol, but not thpearticulate fraction, of lysed human erythrocytes
Summary
Human erythrocytescontainedasolublecytosolic hydration of LTA, produces LTB4 [6, 7], a chemokinetic, epoxide hydrolase for stereospecific enzymatic hydram- yotropic, dihydrodiol ((5S,12R)-5,12-dihydroxy-6,14-cistion of leukotriene A, into leukotrieneB4.The enzyme 8,lO-trans-eicosatetraenoicacid) implicated in inflammatory was purified 1 lOO-foId, to apparent electrophoretic and respiratory disorders [8,9,10].In the absence of epoxide homogeneity, by conventional DEAE-SephaceFfrac- hydrolase, spontaneous, nonenzymatic hydration of the untionationfollowedbyhighperformanceanion ex- stable substrate (LTA4)occurs. Determination of Enzyme Activity-Erythrocyte epoxide hydrolase activity was determined by measuring the conversion of LTA4 into its enzymatic hydration product, LTB4. The material that bound to the column, including hemoglobin and epoxide hydrolase, was eluted at 0.5 ml/ min with a linear gradient of 250 ml each of 0.01 M Tris, pH8.0, and 0.01 M Tris, pH8.0, containing 1M NaCl. The absorbance a t 415 and 280 nm was monitored in 10-ml fractions, and their protein content was determined spectrophotometrically [34].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
