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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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Related in: MedlinePlus

The EC-SOD dimers separate into three forms. Human EC-SOD was subjected to non-reduced SDS-PAGE using a 9% acrylamide gel. Glycosylated EC-SOD (-PNGase F) separates into the disulfide-linked dimer and monomers. The monomers are resolved into aEC-SOD and iEC-SOD as previously shown [23]. The dimer migrates as a fuzzy band of ~52 kDa. When EC-SOD is deglycosylated (+PNGase F) the size of the monomers and dimer is reduced consistent with the removal of a single glycan in each subunit. The deglycosylated dimer resolved into three closely migrating bands of 45 – 49 kDa denoted α, β, and γ as indicated. The position of the deglycosylated monomers is indicated. The gel was stained by Coomassie brilliant blue. A molecular weight marker is indicated on the left. Deglycosylation of EC-SOD improved the separation in non-reduced SDS-PAGE and revealed that the disulfide-bonded dimer migrates as 3 bands most likely caused by differences in folding.
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Figure 1: The EC-SOD dimers separate into three forms. Human EC-SOD was subjected to non-reduced SDS-PAGE using a 9% acrylamide gel. Glycosylated EC-SOD (-PNGase F) separates into the disulfide-linked dimer and monomers. The monomers are resolved into aEC-SOD and iEC-SOD as previously shown [23]. The dimer migrates as a fuzzy band of ~52 kDa. When EC-SOD is deglycosylated (+PNGase F) the size of the monomers and dimer is reduced consistent with the removal of a single glycan in each subunit. The deglycosylated dimer resolved into three closely migrating bands of 45 – 49 kDa denoted α, β, and γ as indicated. The position of the deglycosylated monomers is indicated. The gel was stained by Coomassie brilliant blue. A molecular weight marker is indicated on the left. Deglycosylation of EC-SOD improved the separation in non-reduced SDS-PAGE and revealed that the disulfide-bonded dimer migrates as 3 bands most likely caused by differences in folding.

Mentions: The C-terminally cleaved EC-SOD subunit is lacking the inter-chain disulfide bond and is easily separated from the intact disulfide-bonded EC-SOD dimer during non-reducing SDS-PAGE. We have previously analyzed the C-terminally cleaved EC-SOD subunit and shown the two folding variants are resolved when using optimal electrophoretic conditions [23] (Figure 1). However, the disulfide-bonded EC-SOD dimer remained poorly resolved and migrated as a broad band (Figure 1). Although the N-linked glycan at Asn89 is homogenous [16,27] deglycosylation significantly improved the resolution in unreduced SDS-PAGE resulting in two closely spaced bands that we denoted α and β, and a slightly slower migrating band called γ (Figure 1). Similarly, the folding variants of the deglycosylated C-terminally cleaved EC-SOD subunit was better resolved by SDS-PAGE. This result shows that the EC-SOD dimer exists in three distinct populations which can be separated by SDS-PAGE following deglycosylation.


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 separate into three forms. Human EC-SOD was subjected to non-reduced SDS-PAGE using a 9% acrylamide gel. Glycosylated EC-SOD (-PNGase F) separates into the disulfide-linked dimer and monomers. The monomers are resolved into aEC-SOD and iEC-SOD as previously shown [23]. The dimer migrates as a fuzzy band of ~52 kDa. When EC-SOD is deglycosylated (+PNGase F) the size of the monomers and dimer is reduced consistent with the removal of a single glycan in each subunit. The deglycosylated dimer resolved into three closely migrating bands of 45 – 49 kDa denoted α, β, and γ as indicated. The position of the deglycosylated monomers is indicated. The gel was stained by Coomassie brilliant blue. A molecular weight marker is indicated on the left. Deglycosylation of EC-SOD improved the separation in non-reduced SDS-PAGE and revealed that the disulfide-bonded dimer migrates as 3 bands most likely caused by differences in folding.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The EC-SOD dimers separate into three forms. Human EC-SOD was subjected to non-reduced SDS-PAGE using a 9% acrylamide gel. Glycosylated EC-SOD (-PNGase F) separates into the disulfide-linked dimer and monomers. The monomers are resolved into aEC-SOD and iEC-SOD as previously shown [23]. The dimer migrates as a fuzzy band of ~52 kDa. When EC-SOD is deglycosylated (+PNGase F) the size of the monomers and dimer is reduced consistent with the removal of a single glycan in each subunit. The deglycosylated dimer resolved into three closely migrating bands of 45 – 49 kDa denoted α, β, and γ as indicated. The position of the deglycosylated monomers is indicated. The gel was stained by Coomassie brilliant blue. A molecular weight marker is indicated on the left. Deglycosylation of EC-SOD improved the separation in non-reduced SDS-PAGE and revealed that the disulfide-bonded dimer migrates as 3 bands most likely caused by differences in folding.
Mentions: The C-terminally cleaved EC-SOD subunit is lacking the inter-chain disulfide bond and is easily separated from the intact disulfide-bonded EC-SOD dimer during non-reducing SDS-PAGE. We have previously analyzed the C-terminally cleaved EC-SOD subunit and shown the two folding variants are resolved when using optimal electrophoretic conditions [23] (Figure 1). However, the disulfide-bonded EC-SOD dimer remained poorly resolved and migrated as a broad band (Figure 1). Although the N-linked glycan at Asn89 is homogenous [16,27] deglycosylation significantly improved the resolution in unreduced SDS-PAGE resulting in two closely spaced bands that we denoted α and β, and a slightly slower migrating band called γ (Figure 1). Similarly, the folding variants of the deglycosylated C-terminally cleaved EC-SOD subunit was better resolved by SDS-PAGE. This result shows that the EC-SOD dimer exists in three distinct populations which can be separated by SDS-PAGE following deglycosylation.

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