The interaction of acrylamide with glyceraldehyde-3-phosphate dehydrogenase. Structural modifications in the enzyme studied by fluorescence techniques.
The interaction of acrylamide with rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been investigated in Tris buffer, pH 7.5. When GPDH containing about 1 mol NAD per mol of tetramer is incubated with acrylamide (0.01-0.1 M), the tryptophan emission of GPDH, initially quenched by acrylamide, slowly increases to a value exceeding that recorded before the addition of acrylamide. This effect is not observed in apoenzyme solutions, indicating that the enhancement of fluorescence results from the dissociation of some NAD from the acrylamide treated GPDH. Acrylamide inactivates GPDH but 1 mM NAD protects the enzyme from inactivation. The addition of acrylamide to GPDH, labeled with fluorescein-5-isothiocyanate (GPDH-FITC) increases the fluorescence and decreases the polarization of fluorescein. The fluorescent sulfhydryl reagent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine induces similar changes in the fluorescence properties of GPDH-FITC. This reagent, however, fails to react with GPDH preincubated with acrylamide and the titration of acrylamide treated GPDH with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) indicates the loss of up to 7 cysteine residues per tetramer. Acrylamide also decreases the heat stability of GPDH. Altogether, the data indicate that acrylamide covalently reacts with the active site cysteine residues of GPDH and subsequently induces a conformational change in the enzyme.
Orstan
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Institute of Gerontology, University of Michigan, Ann Arbor 48109-2007.
Gafni
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D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology*; D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D020106:Acrylamide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases