Modification of aldose reductase by S-nitrosoglutathione

Animesh Chandra, Sanjay Srivastava, J. Mark Petrash, Aruni Bhatnagar, Satish Srivastava

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 μM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 ± 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in K(m) for NADPH and K(m) for DL- glyceraldehyde, whereas the K(m) for the lipid peroxidation product, 4- hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP-(H)-binding site of the enzyme.

Original languageEnglish (US)
Pages (from-to)15801-15809
Number of pages9
JournalBiochemistry
Volume36
Issue number50
DOIs
StatePublished - Dec 16 1997

Fingerprint

S-Nitrosoglutathione
Aldehyde Reductase
Enzymes
NADP
Molecular mass
Glutathione
Glyceraldehyde
Electrospray ionization
Glutathione Disulfide
Enzyme activity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chandra, A., Srivastava, S., Petrash, J. M., Bhatnagar, A., & Srivastava, S. (1997). Modification of aldose reductase by S-nitrosoglutathione. Biochemistry, 36(50), 15801-15809. https://doi.org/10.1021/bi9714722

Modification of aldose reductase by S-nitrosoglutathione. / Chandra, Animesh; Srivastava, Sanjay; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Satish.

In: Biochemistry, Vol. 36, No. 50, 16.12.1997, p. 15801-15809.

Research output: Contribution to journalArticle

Chandra, A, Srivastava, S, Petrash, JM, Bhatnagar, A & Srivastava, S 1997, 'Modification of aldose reductase by S-nitrosoglutathione', Biochemistry, vol. 36, no. 50, pp. 15801-15809. https://doi.org/10.1021/bi9714722
Chandra A, Srivastava S, Petrash JM, Bhatnagar A, Srivastava S. Modification of aldose reductase by S-nitrosoglutathione. Biochemistry. 1997 Dec 16;36(50):15801-15809. https://doi.org/10.1021/bi9714722
Chandra, Animesh ; Srivastava, Sanjay ; Petrash, J. Mark ; Bhatnagar, Aruni ; Srivastava, Satish. / Modification of aldose reductase by S-nitrosoglutathione. In: Biochemistry. 1997 ; Vol. 36, No. 50. pp. 15801-15809.
@article{a6819c24ffa147caaaeae76c745194f8,
title = "Modification of aldose reductase by S-nitrosoglutathione",
abstract = "Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 μM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 ± 0.009 M-1 min-1. However, upon exhaustive modification, 30-40{\%} of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in K(m) for NADPH and K(m) for DL- glyceraldehyde, whereas the K(m) for the lipid peroxidation product, 4- hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70{\%} of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30{\%} of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP-(H)-binding site of the enzyme.",
author = "Animesh Chandra and Sanjay Srivastava and Petrash, {J. Mark} and Aruni Bhatnagar and Satish Srivastava",
year = "1997",
month = "12",
day = "16",
doi = "10.1021/bi9714722",
language = "English (US)",
volume = "36",
pages = "15801--15809",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "50",

}

TY - JOUR

T1 - Modification of aldose reductase by S-nitrosoglutathione

AU - Chandra, Animesh

AU - Srivastava, Sanjay

AU - Petrash, J. Mark

AU - Bhatnagar, Aruni

AU - Srivastava, Satish

PY - 1997/12/16

Y1 - 1997/12/16

N2 - Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 μM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 ± 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in K(m) for NADPH and K(m) for DL- glyceraldehyde, whereas the K(m) for the lipid peroxidation product, 4- hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP-(H)-binding site of the enzyme.

AB - Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 μM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 ± 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in K(m) for NADPH and K(m) for DL- glyceraldehyde, whereas the K(m) for the lipid peroxidation product, 4- hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP-(H)-binding site of the enzyme.

UR - http://www.scopus.com/inward/record.url?scp=0031458341&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031458341&partnerID=8YFLogxK

U2 - 10.1021/bi9714722

DO - 10.1021/bi9714722

M3 - Article

C2 - 9398310

AN - SCOPUS:0031458341

VL - 36

SP - 15801

EP - 15809

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 50

ER -