Expression and induction of an immunochemically related class of glutathione S-transferases in Daphnia magna.
The cytosolic glutathione S-transferases (GSTs) are dimeric enzymes that are responsible for the conjugation of glutathione to an electrophilic center of a variety of lipophilic compounds. The purpose of the present study was to characterize the GSTs of Daphnia magna with respect to enzyme multiplicity, molecular weight, isoelectric points, and immunochemical distinction and to determine the inducibility of these enzymes by the prototypical mammalian GST inducer, phenobarbital. GSTs were purified from crude cystosols prepared from daphnids by glutathione-sepharose affinity chromatography. SDS-polyacrylamide gel electrophoresis of the affinity purified GSTs revealed the presence of multiple subunits with molecular weights ranging from 26.9 to 30.2 kDa. Preparative electrofocusing separated GST activity into three major fractions having approximate isoelectric points of 4.5, 4.8 and 5.6. All of the catalytically active fractions contained a single protein band of the same molecular weight (30.2 kDa) during SDS-PAGE. A monoclonal antibody, prepared against the affinity-purified GST proteins, recognized three distinct proteins separated during analytical-scale isoelectric focusing (pI 4.6, 4.7 and 4.8). These proteins may represent a class of GSTs distinct from the GST having a pI of 5.6. Treatment of daphnids with phenobarbital elevated both GST catalytic activity and immunodetectable protein. These results demonstrate that multiple immunochemically related proteins of the same molecular weight but varying isoelectric points are responsible for most of the GST catalytic activity with the substrate 1-chloro-2,4-dinitrobenzene.
Baldwin
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WS
Department of Toxicology, North Carolina State University, Raleigh 27695, USA.
LeBlanc
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GA
D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D003621:Daphnia / Q000201:enzymology*; D004790:Enzyme Induction / Q000187:drug effects; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D007700:Kinetics; D010634:Phenobarbital / Q000494:pharmacology
D007527:Isoenzymes; D005982:Glutathione Transferase; D010634:Phenobarbital