An in vivo and in vitro study of the biochemical characteristics of coal ash-induced epoxide hydrolase in liver microsomes and cytosol of fundulus grandis, 1986

Abstract

Charcoal ash-induced microsomal and cytosolic epoxide hydrolase(s) from liver tissue fractions of an estuarine fish, Fundulus grandis, were isolated and purified by ion-exchange chromatography using Diethylamino-Ethyl Cellulose (DEAE) and Carboxymethyl Cellulose (CM52). Electrophoretic resolution of the purified microsomal and cytosolic epoxide hydrolase(s) from these columns on 10% sodium dodecyl sulphate polyacrylamide slab gels indicated the presence of a liver cytosolic and two microsomal forms of this enzyme in Fundulus grandis. The two forms of microsomal epoxide hydrolase(s) and the cytosolic form, based on their relative mobilities on 10% SDS-polyacrylamide gels, are 49,000 and 59,000 daltons. In vitro translation with membrane-bound and free polysomes from this fish was performed in a cell-free rabbit reticulocyte lysate system. The synthesized epoxide hydrolase(s) were immunoprecipitated with rabbit anti-fish epoxide hydrolase antibody and the products resolved on 10% SDS-polyacrylamide gels. Our findings, based on autoradiographs of these gels, indicate that a cytosolic and two microsomal forms of epoxide hydrolase were, putatively, synthesized in vitro. Co-translational addition of dog pancreatic microsomal membrane to the translation mixture containing free polysomes showed no shift in molecular weight of the synthesized protein, thus, indicating that epoxide hydrolase(s) are not processed via signal sequence cleavage. Post-translational proteolytic digestion with trypsin and chymotrypsin revealed that the two microsomal forms are protected by the membrane, whereas, the cytosolic form is not