TY - JOUR
T1 - A rapid radiometric assay for mammalian cytosolic epoxide hydrolase
AU - Mullin, Christopher A.
AU - Hammock, Bruce D.
N1 - Funding Information:
The authors wish to thank K. Ota and L. Hasegawa of this division and A. Sylwester formerly of this division for their helpful comments throughout this study. This work was supported, in part, by Grant 5-ROl-ES01260-03 from the U. S. Public Health Service and the California Cancer Research Co-ordinating Committee. B. D. Hammock was supported by NIEHS Research Career Development Award 1 K04 ES00046-01.
PY - 1980
Y1 - 1980
N2 - A rapid and sensitive radiometric assay for cytosolic epoxide hydrolase is described. The assay is based on the highly efficient partitioning of unreacted 1-phenyl-1,2-epoxybutane (β-ethylstyrene oxide) from an aqueous mix into isooctane, with the product diol being retained in the aqueous phase. The trans-epoxide is an excellent substrate for both the glutathione transferase and epoxide hydrolase present in mouse liver cytosol, and both enzymes may be monitored simultaneously after appropriate modifications of the assay are made. Also, trans-β-ethylstyrene oxide is hydrated much faster than the cis-isomer in the hepatic cytosol of three different mammalian species, and both isomers are poor substrates for the microsomal epoxide hydrolase. Mouse liver cytosol hydrates the trans-epoxide at 69 nmol/min-mg protein, and a Km of 7.2 × 10−5m is observed. Reduction of 1-phenyl-1-bromo-2-butanone with sodium borotritide and subsequent ring closure of the bromohydrin mix with base was a highly efficient route for radiolabeling β-ethylstyrene oxide. The resulting cis- and trans-epoxides were separable by semipreparative high-performance liquid chromatography.
AB - A rapid and sensitive radiometric assay for cytosolic epoxide hydrolase is described. The assay is based on the highly efficient partitioning of unreacted 1-phenyl-1,2-epoxybutane (β-ethylstyrene oxide) from an aqueous mix into isooctane, with the product diol being retained in the aqueous phase. The trans-epoxide is an excellent substrate for both the glutathione transferase and epoxide hydrolase present in mouse liver cytosol, and both enzymes may be monitored simultaneously after appropriate modifications of the assay are made. Also, trans-β-ethylstyrene oxide is hydrated much faster than the cis-isomer in the hepatic cytosol of three different mammalian species, and both isomers are poor substrates for the microsomal epoxide hydrolase. Mouse liver cytosol hydrates the trans-epoxide at 69 nmol/min-mg protein, and a Km of 7.2 × 10−5m is observed. Reduction of 1-phenyl-1-bromo-2-butanone with sodium borotritide and subsequent ring closure of the bromohydrin mix with base was a highly efficient route for radiolabeling β-ethylstyrene oxide. The resulting cis- and trans-epoxides were separable by semipreparative high-performance liquid chromatography.
UR - http://www.scopus.com/inward/record.url?scp=0019192818&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0019192818&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(80)90551-5
DO - 10.1016/0003-2697(80)90551-5
M3 - Article
C2 - 7447013
AN - SCOPUS:0019192818
SN - 0003-2697
VL - 106
SP - 476
EP - 485
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -