Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the tw...
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PeerJ Inc.
2014-10-01
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| Online Access: | https://peerj.com/articles/634.pdf |
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| author | Ryan A.V. Bell Jeffrey C. Smith Kenneth B. Storey |
| author_facet | Ryan A.V. Bell Jeffrey C. Smith Kenneth B. Storey |
| author_sort | Ryan A.V. Bell |
| collection | DOAJ |
| description | Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40–60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy. |
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| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T08:19:54Z |
| publishDate | 2014-10-01 |
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| spelling | doaj.art-b1a6a59b818047e5b7506e521f7e45bf2023-12-02T21:54:00ZengPeerJ Inc.PeerJ2167-83592014-10-012e63410.7717/peerj.634634Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatusRyan A.V. Bell0Jeffrey C. Smith1Kenneth B. Storey2Department of Chemistry, Carleton University, Ottawa, Ontario, CanadaDepartment of Chemistry, Carleton University, Ottawa, Ontario, CanadaDepartment of Chemistry, Carleton University, Ottawa, Ontario, CanadaGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40–60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.https://peerj.com/articles/634.pdfPosttranslational modificationDifferential scanning fluorimetryTorporMass spectrometryMethylationPhosphorylation |
| spellingShingle | Ryan A.V. Bell Jeffrey C. Smith Kenneth B. Storey Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus PeerJ Posttranslational modification Differential scanning fluorimetry Torpor Mass spectrometry Methylation Phosphorylation |
| title | Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus |
| title_full | Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus |
| title_fullStr | Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus |
| title_full_unstemmed | Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus |
| title_short | Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus |
| title_sort | purification and properties of glyceraldehyde 3 phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel ictidomys tridecemlineatus |
| topic | Posttranslational modification Differential scanning fluorimetry Torpor Mass spectrometry Methylation Phosphorylation |
| url | https://peerj.com/articles/634.pdf |
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