Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress

Heather Smallwood, Nadezhda A. Galeva, Ryan K. Bartlett, Ramona J. Bieber Urbauer, Todd D. Williams, Jeffrey L. Urbauer, Thomas C. Squier

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitrated by peroxynitrite and that methionine side chains do not function as an antioxidant in scavenging peroxynitrite. Thus, separate reactive species are involved in the oxidation of methionine and nitration of Tyr99 whose relative concentrations are determined by solution conditions. The sensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways are sensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker of peroxynitrite-induced changes in cellular function.

Original languageEnglish (US)
Pages (from-to)95-102
Number of pages8
JournalChemical Research in Toxicology
Volume16
Issue number1
DOIs
StatePublished - Jan 1 2003
Externally publishedYes

Fingerprint

Nitration
Oxidative stress
Peroxynitrous Acid
Calmodulin
Oxidative Stress
Methionine
Scavenging
Leucine
Substitution reactions
Oxidation
Oxidants
Nitrates
Mass spectrometry
Tyrosine
Mass Spectrometry
Antioxidants

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

Smallwood, H., Galeva, N. A., Bartlett, R. K., Bieber Urbauer, R. J., Williams, T. D., Urbauer, J. L., & Squier, T. C. (2003). Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress. Chemical Research in Toxicology, 16(1), 95-102. https://doi.org/10.1021/tx025566a

Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress. / Smallwood, Heather; Galeva, Nadezhda A.; Bartlett, Ryan K.; Bieber Urbauer, Ramona J.; Williams, Todd D.; Urbauer, Jeffrey L.; Squier, Thomas C.

In: Chemical Research in Toxicology, Vol. 16, No. 1, 01.01.2003, p. 95-102.

Research output: Contribution to journalArticle

Smallwood, H, Galeva, NA, Bartlett, RK, Bieber Urbauer, RJ, Williams, TD, Urbauer, JL & Squier, TC 2003, 'Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress', Chemical Research in Toxicology, vol. 16, no. 1, pp. 95-102. https://doi.org/10.1021/tx025566a
Smallwood, Heather ; Galeva, Nadezhda A. ; Bartlett, Ryan K. ; Bieber Urbauer, Ramona J. ; Williams, Todd D. ; Urbauer, Jeffrey L. ; Squier, Thomas C. / Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress. In: Chemical Research in Toxicology. 2003 ; Vol. 16, No. 1. pp. 95-102.
@article{8c2a58ce6a274853a1a1619fc6de6628,
title = "Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress",
abstract = "We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitrated by peroxynitrite and that methionine side chains do not function as an antioxidant in scavenging peroxynitrite. Thus, separate reactive species are involved in the oxidation of methionine and nitration of Tyr99 whose relative concentrations are determined by solution conditions. The sensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways are sensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker of peroxynitrite-induced changes in cellular function.",
author = "Heather Smallwood and Galeva, {Nadezhda A.} and Bartlett, {Ryan K.} and {Bieber Urbauer}, {Ramona J.} and Williams, {Todd D.} and Urbauer, {Jeffrey L.} and Squier, {Thomas C.}",
year = "2003",
month = "1",
day = "1",
doi = "10.1021/tx025566a",
language = "English (US)",
volume = "16",
pages = "95--102",
journal = "Chemical Research in Toxicology",
issn = "0893-228X",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress

AU - Smallwood, Heather

AU - Galeva, Nadezhda A.

AU - Bartlett, Ryan K.

AU - Bieber Urbauer, Ramona J.

AU - Williams, Todd D.

AU - Urbauer, Jeffrey L.

AU - Squier, Thomas C.

PY - 2003/1/1

Y1 - 2003/1/1

N2 - We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitrated by peroxynitrite and that methionine side chains do not function as an antioxidant in scavenging peroxynitrite. Thus, separate reactive species are involved in the oxidation of methionine and nitration of Tyr99 whose relative concentrations are determined by solution conditions. The sensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways are sensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker of peroxynitrite-induced changes in cellular function.

AB - We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitrated by peroxynitrite and that methionine side chains do not function as an antioxidant in scavenging peroxynitrite. Thus, separate reactive species are involved in the oxidation of methionine and nitration of Tyr99 whose relative concentrations are determined by solution conditions. The sensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways are sensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker of peroxynitrite-induced changes in cellular function.

UR - http://www.scopus.com/inward/record.url?scp=0037664484&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037664484&partnerID=8YFLogxK

U2 - 10.1021/tx025566a

DO - 10.1021/tx025566a

M3 - Article

VL - 16

SP - 95

EP - 102

JO - Chemical Research in Toxicology

JF - Chemical Research in Toxicology

SN - 0893-228X

IS - 1

ER -