Oxidative modification of glutamine synthetase by 2,2'-azobis(2- amidinopropane) dihydrochloride.
In the present study, we examined the pattern of protein modification elicited by alkylperoxyl radicals and alkylperoxides. To this end, we exposed glutamine synthetase (GS) and the peptide melittin to solutions containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which is known to decompose in aqueous, aerobic solutions to yield alkyl radicals and alkylperoxides. Under our conditions, pH 7.4, 37 degrees C, the AAPH-dependent formation of alkylhydroperoxide increased linearly with time and led to 40% inactivation of GS in 1 h and to complete inactivation in 4 h. Complete inactivation was associated with the loss of 2 of 16 histidine residues, 6 of 17 tyrosine residues, 5 of 16 methionine residues, and all of the tryptophan residues (2 residues) per subunit. Inactivation of GS was associated also with some protein fragmentation and the formation of some higher molecular weight aggregates. Exposure of GS to AAPH led also to the generation of protein carbonyl derivatives (0.34 mol/mol subunit) and to formation of a significant amount (0.038 mol/mol subunits) of quinoprotein derivatives. To investigate the mechanism of tryptophan modification, the 26-amino-acid peptide, melittin, which contains one tryptophan but no histidine, tyrosine, or methionine residues, was treated with AAPH. N-Formylkynurenine was identified as the major product of tryptophan oxidation in melittin.
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Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland, 20892-0342, USA.
Chao
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Stadtman
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D000578:Amidines / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005974:Glutamate-Ammonia Ligase / Q000187:drug effects* / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D007737:Kynurenine / Q000031:analogs & derivatives / Q000378:metabolism; D008555:Melitten / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000578:Amidines; D016877:Oxidants; D017382:Reactive Oxygen Species; C007772:N'-formylkynurenine; D008555:Melitten; D007737:Kynurenine; D014443:Tyrosine; D006639:Histidine; C046728:2,2'-azobis(2-amidinopropane); D014364:Tryptophan; D008715:Methionine; D005974:Glutamate-Ammonia Ligase