Rat and human liver cytosolic epoxide hydrolases: evidence for multiple forms at level of protein and mRNA.
Two forms of human liver cytosolic epoxide hydrolase (cEH) with diagnostic substrate specificity for trans-stilbene oxide (cEHTSO) and cis-stilbene oxide (cEHCSO) have been identified, and cEHCSO was purified to apparent homogeneity. The enzyme had a monomer molecular weight of 49 kDa and an isoelectric point of 9.2. Pure cEHCSO hydrolyzed CSO at a rate of 145 nmole/min/mg. TSO was not metabolized at a detectable level, and like cEHTSO, the enzyme was about three times more active at pH 7.4 than at pH 9.0. Unlike cEHTSO, cEHCSO was efficiently inhibited by 1 mM 1-trichloropropene oxide (90.5%) and 1 mM STO (92%). Similarly, liver cEH purified 541-fold from fenofibrate induced Fischer 344 rats was shown to be a native 120 kDa dimer of two 61 kDa subunits. The enzyme expressed maximum activity of 205 nmole/min/mg at pH 7.4 toward the diagnostic substrate TSO with an apparent Km of 1.7 microM. In Western blots, polyclonal antibodies against rat liver cEH were shown to recognize a single 61 kDa protein band from liver cytosol of rat, mouse, guinea pig, Syrian hamster, and rabbit. This antibody precipitated neither human liver cEHTSO or cEHCSO. Antibodies against rat liver microsomal epoxide hydrolase reacted with cEHCSO in the Western blot and on immunoprecipitation. Using antibodies against rat liver cEH, 24 positive clones were picked upon colony blot screening of a pEX 1/E. coli POP 2136 expression library.(ABSTRACT TRUNCATED AT 250 WORDS)
Thomas
H
H
Institute of Toxicology, University of Mainz, Federal Republic of Germany.
Schladt
L
L
Doehmer
J
J
Knehr
M
M
Oesch
F
F
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004851:Epoxide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007120:Immunochemistry; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013045:Species Specificity
D012333:RNA, Messenger; D004247:DNA; D004851:Epoxide Hydrolases