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Enzymatic characteristics of chimeric mYc/rYc1 glutathione S-transferases
K P Van Ness1, T M Buetler, D L Eaton
1Department of Environmental Health, University of Washington, Seattle 98195.
Abstract:
Mice are resistant to aflatoxin carcinogenicity primarily due to expression of a glutathione S-transferase (mYc) with high catalytic activity toward aflatoxin B1-8,9-epoxide (AFBO). In contrast, rats are more sensitive to aflatoxin carcinogenicity due to the constitutive expression of a glutathione S-transferase with relatively low catalytic activity toward AFBO (rYc1). To identify the contribution of different regions of the mYc protein that confer high catalytic activity toward AFBO, six chimeric mYc/rYc1 GST enzymes were generated utilizing full and partial restriction enzyme digestions at two conserved StyI sites in the mYc and rYc1 complementary DNAs (between amino acid residues 56-57 and 142-143). Recombinant wild-type and chimeric glutathione S-transferases were bacterially expressed, affinity purified, and their catalytic activities measured toward AFBO, delta 5-androstene-3,17-dione, 1-chloro-2,4-dinitrobenzene, and ethacrynic acid. The set of chimeras displayed a wide range of catalytic activities toward the substrates assayed. The chimeras with the greatest activity toward AFBO were 1:56rat-57: 221mouse and 1:56mouse-57:142rat-143:221mouse, with AFBO conjugating activities 200 and 8 times greater than wild-type rYc1, respectively. These results demonstrate that the residues that confer high AFBO conjugation activity in mYc are located in the region spanning residues 57-221.
Insights
Mice resist aflatoxin by expressing glutathione S-transferase (mYc) highly active against aflatoxin epoxide (AFBO). Rat glutathione S-transferase (rYc1) is less active, making rats more sensitive. This study identified key regions in mYc responsible for high AFBO conjugation.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Mice exhibit resistance to aflatoxin carcinogenicity, attributed to a highly active glutathione S-transferase (mYc) against aflatoxin B1-8,9-epoxide (AFBO).
- Rats are more susceptible due to a less catalytically active glutathione S-transferase (rYc1) towards AFBO.
Purpose of the Study:
- To pinpoint the specific regions within the mYc protein that confer high catalytic activity towards AFBO.
- To understand the structural basis of differential aflatoxin sensitivity between mice and rats.
Main Methods:
- Generation of six chimeric glutathione S-transferase (GST) enzymes by combining regions of mYc and rYc1 complementary DNAs at conserved StyI sites (residues 56-57 and 142-143).
- Bacterial expression, affinity purification, and characterization of catalytic activities of wild-type and chimeric GSTs against AFBO and other substrates.
Main Results:
- Chimeric GSTs exhibited varied catalytic activities, with two constructs showing significantly enhanced AFBO conjugation.
- The chimeric enzymes 1:56rat-57:221mouse and 1:56mouse-57:142rat-143:221mouse displayed AFBO conjugating activities 200-fold and 8-fold higher than wild-type rYc1, respectively.
- These findings implicate the amino acid region spanning residues 57-221 of mYc as critical for high AFBO conjugation activity.
Conclusions:
- The region between amino acid residues 57 and 221 in mouse glutathione S-transferase (mYc) is essential for its high catalytic activity against aflatoxin epoxide (AFBO).
- This structural insight contributes to understanding the molecular mechanisms underlying species-specific differences in aflatoxin detoxification and carcinogenicity.