Enzymatic characteristics of chimeric mYc/rYc1 glutathione S-transferases.
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.
Van Ness
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Department of Environmental Health, University of Washington, Seattle 98195.
Buetler
T M
TM
Eaton
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DL
D016604:Aflatoxin B1 / Q000378:metabolism*; D000595:Amino Acid Sequence; D000735:Androstenedione / Q000031:analogs & derivatives / Q000378:metabolism; D004137:Dinitrochlorobenzene / Q000378:metabolism*; D004976:Ethacrynic Acid / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D004137:Dinitrochlorobenzene; D011993:Recombinant Fusion Proteins; D000735:Androstenedione; D016604:Aflatoxin B1; D005982:Glutathione Transferase; D004976:Ethacrynic Acid