Oxidative stress transforms acetylcholinesterase to a molten-globule-like state.
Exposure of purified acetylcholinesterase from Torpedo california to a system generating oxygen radicals (viz. ascorbic acid/Fe(EDTA)2/H2O2) inactivated the enzyme. The enzyme retained its native dimeric form, but electrophoresis under denaturing conditions showed some cleavage of peptide bonds. Spectroscopic characterization revealed a shift to the red in the intrinsic fluorescence emission peak, a large decrease in molar ellipticity in the near UV with a much smaller decrease in the far UV, and increased binding of the amphiphilic probe, 1-anilino-8-naphthalene sulfonate, all relative to native enzyme. The treated enzyme was also highly susceptible to proteolysis. These data show that oxygen radical treatment converts acetylcholinesterase to a partially unfolded state, which retains most of its secondary structure but lacks substantial tertiary structure, thus resembling a 'molten globule' state. This model system may offer a mechanism for explaining the consequences of oxidative stress in vivo: partially unfolded proteins generated by oxidative stress may interact with molecular chaperons of the heat shock family, thus activating the heat-shock factor and, thereby, activating heat-shock genes.
Weiner
L
L
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Kreimer
D
D
Roth
E
E
Silman
I
I
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D004492:Edetic Acid / Q000494:pharmacology; D004557:Electric Organ / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000494:pharmacology; D005609:Free Radicals / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D017510:Protein Folding; D013053:Spectrophotometry; D014101:Torpedo
D005296:Ferrous Compounds; D005609:Free Radicals; D016877:Oxidants; C055526:Fe(II)-EDTA; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D000110:Acetylcholinesterase; D001205:Ascorbic Acid