Ws. Baldwin et Ga. Leblanc, EXPRESSION AND INDUCTION OF ALL IMMUNOCHEMICALLY RELATED CLASS OF GLUTATHIONE S-TRANSFERASES IN DAPHNIA-MAGNA, Comparative biochemistry and physiology. B. Comparative biochemistry, 113(2), 1996, pp. 261-267
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.