EXPRESSION AND INDUCTION OF ALL IMMUNOCHEMICALLY RELATED CLASS OF GLUTATHIONE S-TRANSFERASES IN DAPHNIA-MAGNA

The cytosolic glutathione S-transferases (GSTs) are dimeric enzymes th at are responsible for the conjugation of glutathione to an electrophi lic center of a variety of lipophilic compounds. The purpose of the pr esent study was to characterize the GSTs of Daphnia magna with respect to enzyme multiplicity, molecular weight, isoelectric points, and imm unochemical distinction and to determine the inducibility of these enz ymes by the prototypical mammalian GST inducer, phenobarbital. GSTs we re purified from crude cystosols prepared. from daphnids by glutathion e-sepharose affinity chromatography. SDS-polyacrylamide gel electropho resis oi the affinity purified GSTs revealed the presence of multiple subunits with molecular weights ranging from 26.9 to 30.2 kDa. Prepara tive 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 antibo dy, prepared against the affinity-purified GST proteins, recognized th ree distinct proteins separated during analytical-scale isoelectric fo cusing (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 w ith phenobarbital elevated both GST catalytic activity; and immunodefe ctable protein. These results demonstrate that multiple immunochemical ly related proteins of the same molecular weight but varying isoelectr ic points are responsible for most of the GST catalytic activity with the substrate 1-chloro-2,4-dinitrobenzene.