A role for glutathione in reactions between oxidants and proteins

dc.contributor.advisor James A. Thomas
dc.contributor.author Mallis, Robert
dc.contributor.department Biochemistry, Biophysics and Molecular Biology
dc.date 2018-08-23T03:01:59.000
dc.date.accessioned 2020-06-30T07:32:32Z
dc.date.available 2020-06-30T07:32:32Z
dc.date.copyright Sat Jan 01 00:00:00 UTC 2000
dc.date.issued 2000-01-01
dc.description.abstract <p>This dissertation examines the role of reduced glutathione (GSH) in the oxidation of proteins. Carbonic anhydrase III is a cytosolic protein which is S-glutathiolated in cells under oxidative stress. The pure protein was found here to be S-glutathiolated by two oxidants, 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and hydrogen peroxide (H2O2). At physiologically relevant molar ratios of GSH to protein, GSH was found to mediate protection of carbonic anhydrase III from irreversible oxidation via S-glutathiolation. Both oxidants were found to react directly with the protein. S-glutathiolation. appears to result from the formation of an activated protein thiol that reacts with GSH;H-Ras, a low molecular weight G-protein that regulates proliferation and differentiation in cells, may be regulated by oxidative events. For this reason, oxidative modifications of the cysteine residues of H-Ras were studied. The pure protein was modified on multiple thiols when incubated with thiol oxidants. H2O2 + GSH was found to S-glutathiolate H-Ras on at least one cysteine, while either diamide + GSH or glutathione disulfide (GSSG) was found to S-glutathiolate at least two H-Ras cysteines. The NO donor S-nitrosoglutathione caused S-nitrosylation of H-Ras on four cysteine residues;Within NIH/3T3 cells overexpressing H-Ras, H-Ras was S-glutathiolated on multiple thiols by diamide. At least one of the cysteine residues modified in cells by diamide is normally lipidated, suggesting a role for oxidation in regulating the membrane association of H-Ras. In NIH/3T3 cells overexpressing H-Ras, S-nitrosocysteine was found to cause both S-nitrosylation and S-glutathiolation of H-Ras. Thus, oxidative modification of H-Ras can occur simultaneously on multiple thiols and by multiple mechanisms;The effect of S-nitrosocysteine on low molecular weight thiols and soluble proteins in NIH/3T3 cells was also studied. S-nitrosocysteine was found to be an effective S-nitrosating agent, causing S-nitrosylation of glutathione and proteins. S-nitrosocysteine was also found to be an effective oxidant, causing formation of glutathione disulfide, cysteine-glutathione disulfide, cystine, S-glutathiolated and S-cysteylated proteins. The biological effects of S-nitrosocysteine are likely to be mediated not only through nitrosative, but also oxidative events. Thus cellular GSH pools interact significantly with S-nitrosocysteine and may be a factor in cell-specific variations in responses to S-nitrosothiols.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/rtd/13916/
dc.identifier.articleid 14915
dc.identifier.contextkey 6950763
dc.identifier.doi https://doi.org/10.31274/rtd-180813-15270
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath rtd/13916
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/67442
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/rtd/13916/r_9962832.pdf|||Fri Jan 14 20:04:10 UTC 2022
dc.subject.disciplines Biochemistry
dc.subject.keywords Biochemistry
dc.subject.keywords biophysics
dc.subject.keywords and molecular biology
dc.subject.keywords Biochemistry
dc.title A role for glutathione in reactions between oxidants and proteins
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication faf0a6cb-16ca-421c-8f48-9fbbd7bc3747
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
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