Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation

David C. Salo, Robert E. Pacifici, Sharon W. Lin, Cecilia R Giulivi, Kelvin J A Davies

Research output: Contribution to journalArticle

223 Citations (Scopus)

Abstract

Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cupro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O- 2 to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50%) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O- 2 and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75%) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O- 2, and H2O2 + O- 2 treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a "Fraction II" of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD. In reticulocytes (and perhaps other cells which overexpress SOD as part of an oxidative stress response to O- 2 or H2O2) macroxyproteinase may form part of a closed-loop system for SOD turnover, regulated by the flux of O- 2 (or H2O2) to which SOD is exposed.

Original languageEnglish (US)
Pages (from-to)11919-11927
Number of pages9
JournalJournal of Biological Chemistry
Volume265
Issue number20
StatePublished - Jul 15 1990
Externally publishedYes

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Proteolysis
Superoxide Dismutase
Blood
Erythrocytes
Copper
Proteasome Endopeptidase Complex
Enzymes
Xanthine
Oxidative stress
Xanthine Oxidase
Serine
Zinc
Reticulocytes
Peptide Hydrolases
Adenosine Triphosphate
Cells
Rabbits
Cell Extracts
Sulfhydryl Reagents
Denaturation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. / Salo, David C.; Pacifici, Robert E.; Lin, Sharon W.; Giulivi, Cecilia R; Davies, Kelvin J A.

In: Journal of Biological Chemistry, Vol. 265, No. 20, 15.07.1990, p. 11919-11927.

Research output: Contribution to journalArticle

Salo, David C. ; Pacifici, Robert E. ; Lin, Sharon W. ; Giulivi, Cecilia R ; Davies, Kelvin J A. / Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 20. pp. 11919-11927.
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abstract = "Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cupro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O- 2 to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50{\%}) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O- 2 and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75{\%}) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O- 2, and H2O2 + O- 2 treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a {"}Fraction II{"} of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD. In reticulocytes (and perhaps other cells which overexpress SOD as part of an oxidative stress response to O- 2 or H2O2) macroxyproteinase may form part of a closed-loop system for SOD turnover, regulated by the flux of O- 2 (or H2O2) to which SOD is exposed.",
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T1 - Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation

AU - Salo, David C.

AU - Pacifici, Robert E.

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N2 - Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cupro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O- 2 to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50%) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O- 2 and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75%) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O- 2, and H2O2 + O- 2 treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a "Fraction II" of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD. In reticulocytes (and perhaps other cells which overexpress SOD as part of an oxidative stress response to O- 2 or H2O2) macroxyproteinase may form part of a closed-loop system for SOD turnover, regulated by the flux of O- 2 (or H2O2) to which SOD is exposed.

AB - Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cupro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O- 2 to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50%) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O- 2 and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75%) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O- 2, and H2O2 + O- 2 treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a "Fraction II" of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD. In reticulocytes (and perhaps other cells which overexpress SOD as part of an oxidative stress response to O- 2 or H2O2) macroxyproteinase may form part of a closed-loop system for SOD turnover, regulated by the flux of O- 2 (or H2O2) to which SOD is exposed.

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