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The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulates enzymatic activity.

Petersen SV, Valnickova Z, Oury TD, Crapo JD, Chr Nielsen N, Enghild JJ - BMC Biochem. (2007)

Bottom Line: The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai).This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity.This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO) Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. svp@mb.au.dk

ABSTRACT

Background: Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875-80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist.

Results: The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits.

Conclusion: This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.

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The EC-SOD dimers can be separated by reverse-phase HPLC. EC-SOD can be separated into monomer and dimer by reverse-phase HPLC [23]. The isolated dimer was subsequently denatured and alkylated using iodoacetamide. The alkylated material was subjected to reverse-phase HPLC using a C8 column. The material separated into three major peaks without base-line separation. The collected fractions were denoted F1, F2, and F3 as indicated. The material was subsequently analyzed by non-reducing SDS-PAGE using a 9% acrylamide gel (inset). Protein was visualized by silver staining. Three distinct bands were detected and denoted α, β, and γ according to figure 1. A molecular weight marker is indicated on the left.
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Figure 2: The EC-SOD dimers can be separated by reverse-phase HPLC. EC-SOD can be separated into monomer and dimer by reverse-phase HPLC [23]. The isolated dimer was subsequently denatured and alkylated using iodoacetamide. The alkylated material was subjected to reverse-phase HPLC using a C8 column. The material separated into three major peaks without base-line separation. The collected fractions were denoted F1, F2, and F3 as indicated. The material was subsequently analyzed by non-reducing SDS-PAGE using a 9% acrylamide gel (inset). Protein was visualized by silver staining. Three distinct bands were detected and denoted α, β, and γ according to figure 1. A molecular weight marker is indicated on the left.

Mentions: The C-terminally cleaved EC-SOD subunit and disulfide-linked dimer can be separated by reverse-phase HPLC [23]. Moreover, the C-terminally cleaved EC-SOD subunit can be further separated into the two different folding variants by reverse-phase HPLC following denaturation and S-carboxyamidomethylation [23]. To investigate whether the three electrophoretic variants of dimeric EC-SOD observed by SDS-PAGE could similarly be resolved, we subjected alkylated dimeric EC-SOD to reverse-phase analysis using an octyl-derivatized solid support. The material was found to separate into three major peaks without baseline separation (Figure 2) and fractions were collected manually to reduce cross contamination and following subjected to SDS-PAGE analysis (Figure 2, inset). This analysis revealed that fraction 1 contained components α and β; fraction 2 contained components β and γ; and fraction 3 was found to contain component γ. Based on this pattern of separation, it is likely that baseline separation would have produced three distinct fractions containing isolated components. We conclude that the 3 disulfide-bonded EC-SOD variants designated α, β and γ can be separated by reverse-phase HPLC and that these, based on the difference in electrophoretic mobility, represent folding variants.


The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulates enzymatic activity.

Petersen SV, Valnickova Z, Oury TD, Crapo JD, Chr Nielsen N, Enghild JJ - BMC Biochem. (2007)

The EC-SOD dimers can be separated by reverse-phase HPLC. EC-SOD can be separated into monomer and dimer by reverse-phase HPLC [23]. The isolated dimer was subsequently denatured and alkylated using iodoacetamide. The alkylated material was subjected to reverse-phase HPLC using a C8 column. The material separated into three major peaks without base-line separation. The collected fractions were denoted F1, F2, and F3 as indicated. The material was subsequently analyzed by non-reducing SDS-PAGE using a 9% acrylamide gel (inset). Protein was visualized by silver staining. Three distinct bands were detected and denoted α, β, and γ according to figure 1. A molecular weight marker is indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100054&req=5

Figure 2: The EC-SOD dimers can be separated by reverse-phase HPLC. EC-SOD can be separated into monomer and dimer by reverse-phase HPLC [23]. The isolated dimer was subsequently denatured and alkylated using iodoacetamide. The alkylated material was subjected to reverse-phase HPLC using a C8 column. The material separated into three major peaks without base-line separation. The collected fractions were denoted F1, F2, and F3 as indicated. The material was subsequently analyzed by non-reducing SDS-PAGE using a 9% acrylamide gel (inset). Protein was visualized by silver staining. Three distinct bands were detected and denoted α, β, and γ according to figure 1. A molecular weight marker is indicated on the left.
Mentions: The C-terminally cleaved EC-SOD subunit and disulfide-linked dimer can be separated by reverse-phase HPLC [23]. Moreover, the C-terminally cleaved EC-SOD subunit can be further separated into the two different folding variants by reverse-phase HPLC following denaturation and S-carboxyamidomethylation [23]. To investigate whether the three electrophoretic variants of dimeric EC-SOD observed by SDS-PAGE could similarly be resolved, we subjected alkylated dimeric EC-SOD to reverse-phase analysis using an octyl-derivatized solid support. The material was found to separate into three major peaks without baseline separation (Figure 2) and fractions were collected manually to reduce cross contamination and following subjected to SDS-PAGE analysis (Figure 2, inset). This analysis revealed that fraction 1 contained components α and β; fraction 2 contained components β and γ; and fraction 3 was found to contain component γ. Based on this pattern of separation, it is likely that baseline separation would have produced three distinct fractions containing isolated components. We conclude that the 3 disulfide-bonded EC-SOD variants designated α, β and γ can be separated by reverse-phase HPLC and that these, based on the difference in electrophoretic mobility, represent folding variants.

Bottom Line: The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai).This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity.This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO) Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. svp@mb.au.dk

ABSTRACT

Background: Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875-80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist.

Results: The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits.

Conclusion: This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.

Show MeSH
Related in: MedlinePlus