Matches in SemOpenAlex for { <https://semopenalex.org/work/W1799372645> ?p ?o ?g. }
- W1799372645 abstract "Reactive oxygen species (ROS) are formed during normal respiration in the mitochondria through electron leakage to oxygen. During normal metabolism, several enzymes and low-molecular weight antioxidants work towards eliminating ROS in a well-coordinated manner. The tripeptide glutathione is an integral part of this antioxidant network, and functions both as a reducing factor and as a substrate by several enzymes. When the antioxidant defence of the cell is overwhelmed, “oxidative stress” develops, and cellular redox systems become oxidised. Oxidative stress can result through metabolism of foreign compounds, ischemia-reperfusion, or exposure to UV-radiation, and may also develop naturally during aging. Glutathione (GSH) has long been known to reversibly bind cellular proteins, particularly during oxidative stress, in a reaction catalysed by glutaredoxin, previously known as thioltransferase. These modified protein substrates include several metabolic enzymes and cytoskeletal proteins, which may be thus functionally regulated as a cellular response to stress. However, a comparison of the constitutive and the stressed situations is difficult, since the current analysis protocols for high-throughput analysis are not sensitive enough to identify low level glutathionylation during constitutive metabolism. Hence, there is interest in developing more sensitive methods to accurately portray glutathionylation patterns in the cell. A method was developed based on the specific reduction of glutathione-protein mixed disulphides by glutaredoxin, the reaction of liberated protein thiols with Nethylmaleimide-biotin, affinity purification of tagged proteins, and identification by twodimensional gel electrophoresis and mass spectrometry. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These included protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. The method developed in this study is high-throughput, and more importantly, can specifically identify S-glutathionylated proteins from cells – both stressed cells and cells undergoing constitutive metabolism – with minimal disruption of cellular function. Therefore, the method can successively be used to study posttranslational redox modification of cellular proteins – a potentially significant biochemical control mechanism in coordinating cellular function. The protocol was further developed to allow for affinity purification and analysis of trypsinised S-glutathionylated proteins under non-reducing conditions, in an attempt to avoid the cumbersome 2-D gel electrophoresis step and at the same time identify the sites of S-glutathionylation by tandem mass spectrometry. The glutathionylation sites of gamma-actin, heat-shock protein 70 and elongation factor 1-1 were identified in this way, but the method was hampered by unspecific binding of peptides to the avidinaffinity column, a problem also encountered in other studies. In this study, for the first time, a method has been developed for high-throughput identification of S-glutathionylated proteins without interference with cellular function. This protocol has also been modified to include identification of the S-glutathionylation sites of the proteins involved, although difficulties in peptide affinity capture interfere with specificity and high-throughput analysis. © Ylva Pamment, 2005 ISBN 91-7140-248-9 To James, and to our unborn baby" @default.
- W1799372645 created "2016-06-24" @default.
- W1799372645 creator A5082650495 @default.
- W1799372645 date "2005-02-21" @default.
- W1799372645 modified "2023-09-24" @default.
- W1799372645 title "Novel methods for the identification of cellular S-glutathionylated proteins and sites of glutathionedependent modification using affinity chromatography and proteomic analyses" @default.
- W1799372645 cites W1027091479 @default.
- W1799372645 cites W1497947681 @default.
- W1799372645 cites W1498520518 @default.
- W1799372645 cites W1501442799 @default.
- W1799372645 cites W1511013913 @default.
- W1799372645 cites W1512686244 @default.
- W1799372645 cites W1535785663 @default.
- W1799372645 cites W1537037928 @default.
- W1799372645 cites W1563653502 @default.
- W1799372645 cites W1574557424 @default.
- W1799372645 cites W1576348820 @default.
- W1799372645 cites W1591945630 @default.
- W1799372645 cites W1786911182 @default.
- W1799372645 cites W183647143 @default.
- W1799372645 cites W1864853792 @default.
- W1799372645 cites W1891967301 @default.
- W1799372645 cites W1949043027 @default.
- W1799372645 cites W1966221856 @default.
- W1799372645 cites W1967487924 @default.
- W1799372645 cites W1969416735 @default.
- W1799372645 cites W1971268569 @default.
- W1799372645 cites W1971677443 @default.
- W1799372645 cites W1971741567 @default.
- W1799372645 cites W1972400891 @default.
- W1799372645 cites W1973328612 @default.
- W1799372645 cites W1973475159 @default.
- W1799372645 cites W1973554224 @default.
- W1799372645 cites W1973958759 @default.
- W1799372645 cites W1974970141 @default.
- W1799372645 cites W1976896825 @default.
- W1799372645 cites W1976976084 @default.
- W1799372645 cites W1977229794 @default.
- W1799372645 cites W1978237064 @default.
- W1799372645 cites W1982760825 @default.
- W1799372645 cites W1986956461 @default.
- W1799372645 cites W1987325894 @default.
- W1799372645 cites W1989953524 @default.
- W1799372645 cites W1990201609 @default.
- W1799372645 cites W1990798651 @default.
- W1799372645 cites W1990851262 @default.
- W1799372645 cites W1991108231 @default.
- W1799372645 cites W1991905599 @default.
- W1799372645 cites W1992390260 @default.
- W1799372645 cites W1996053335 @default.
- W1799372645 cites W1997898173 @default.
- W1799372645 cites W2002078917 @default.
- W1799372645 cites W2002409083 @default.
- W1799372645 cites W2002794726 @default.
- W1799372645 cites W2003055213 @default.
- W1799372645 cites W2005904769 @default.
- W1799372645 cites W2009082798 @default.
- W1799372645 cites W2009588836 @default.
- W1799372645 cites W2010094123 @default.
- W1799372645 cites W2013607754 @default.
- W1799372645 cites W2013660147 @default.
- W1799372645 cites W2015732856 @default.
- W1799372645 cites W2021242983 @default.
- W1799372645 cites W2022183036 @default.
- W1799372645 cites W2025532989 @default.
- W1799372645 cites W2032738414 @default.
- W1799372645 cites W2035554488 @default.
- W1799372645 cites W2038349917 @default.
- W1799372645 cites W2040706334 @default.
- W1799372645 cites W2043353183 @default.
- W1799372645 cites W2043817490 @default.
- W1799372645 cites W2044881755 @default.
- W1799372645 cites W2049821479 @default.
- W1799372645 cites W2056115953 @default.
- W1799372645 cites W2060833911 @default.
- W1799372645 cites W2063205422 @default.
- W1799372645 cites W2065463296 @default.
- W1799372645 cites W2067351147 @default.
- W1799372645 cites W2068001141 @default.
- W1799372645 cites W2068861782 @default.
- W1799372645 cites W2070693463 @default.
- W1799372645 cites W2070693959 @default.
- W1799372645 cites W2072265495 @default.
- W1799372645 cites W2084518663 @default.
- W1799372645 cites W2085097402 @default.
- W1799372645 cites W2086064567 @default.
- W1799372645 cites W2088054578 @default.
- W1799372645 cites W2089379767 @default.
- W1799372645 cites W2092932922 @default.
- W1799372645 cites W2094068195 @default.
- W1799372645 cites W2097463865 @default.
- W1799372645 cites W2101942650 @default.
- W1799372645 cites W2102305410 @default.
- W1799372645 cites W2106762735 @default.
- W1799372645 cites W2111208057 @default.
- W1799372645 cites W2124940471 @default.
- W1799372645 cites W2141819645 @default.
- W1799372645 cites W2146890885 @default.
- W1799372645 cites W2149668250 @default.
- W1799372645 cites W2160219722 @default.