Structural studies on human glutathione S-transferase pi. Family of native-specific monoclonal antibodies used to block catalysis.
The glutathione S-transferases are a family of related detoxification enzymes that have been shown to conjugate numerous electrophiles to the common cellular thiol glutathione. We have generated a panel of monoclonal antibodies against the human pi class isozyme of this enzyme, and, in this report, we characterize the binding of these antibodies to the glutathione S-transferase antigen. Of the 10 monoclonal antibodies that we have isolated, 7 are able to recognize the native form of the enzyme while the remaining 3 are only able to bind to glutathione S-transferase pi in assays that partially denature the antigen, such as an enzyme-linked immunosorbent assay or a Western blot. We synthesized seven partial protein fragments and asked whether the monoclonal antibodies could bind to these fragments in an immunoprecipitation reaction. The antibodies that can bind the native form of the enzyme all bind to the carboxyl-terminal domain of the protein. Two antibodies are able to inhibit the glutathione S-transferase-catalyzed reaction noncompetitively against glutathione. Incubation of a 10-fold molar excess of either antibody over enzyme can inhibit the reaction by 50%. We have also used the same protein fragments of glutathione S-transferase pi to show that amino acids 1-77 retain the capacity to bind glutathione in a glutathione-agarose binding assay.
Gulick
A M
AM
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Goihl
A L
AL
Fahl
W E
WE
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002384:Catalysis; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011489:Protein Denaturation
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D004247:DNA; D005982:Glutathione Transferase