Abstract
Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 183-190 |
| Number of pages | 8 |
| Journal | Analytical Biochemistry |
| Volume | 297 |
| Issue number | 2 |
| DOIs | |
| State | Published - Oct 15 2001 |
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Keywords
- GlutathioneS-transferase
- In-flammation
- Myeloperoxidase
- Nitric oxide
- Peroxynitrite
- Protein oxidation
- Tyrosine nitration
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants : Exploring a role for tyrosine nitration. / Wong, Patrick S Y; Eiserich, Jason P.; Reddy, Sharanya; Lopez, C. Leticia; Cross, Carroll E; van der VlieT, Albert.
In: Analytical Biochemistry, Vol. 297, No. 2, 15.10.2001, p. 183-190.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants
T2 - Exploring a role for tyrosine nitration
AU - Wong, Patrick S Y
AU - Eiserich, Jason P.
AU - Reddy, Sharanya
AU - Lopez, C. Leticia
AU - Cross, Carroll E
AU - van der VlieT, Albert
PY - 2001/10/15
Y1 - 2001/10/15
N2 - Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.
AB - Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.
KW - GlutathioneS-transferase
KW - In-flammation
KW - Myeloperoxidase
KW - Nitric oxide
KW - Peroxynitrite
KW - Protein oxidation
KW - Tyrosine nitration
UR - http://www.scopus.com/inward/record.url?scp=85007624583&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85007624583&partnerID=8YFLogxK
U2 - 10.1006/abio.2001.5325
DO - 10.1006/abio.2001.5325
M3 - Article
AN - SCOPUS:85007624583
VL - 297
SP - 183
EP - 190
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -