Abstract
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.
Highlights
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is primarily present within the cell as a homotetramer, comprised of approximately 37 kDa subunits
While known to exist as a single isomer, GAPDH has been found to be extensively modified by posttranslational modifications
Purification of GAPDH GAPDH from skeletal muscle of 13-lined ground was purified using a Blue-agarose affinity column followed by a hydrophobic column, phenyl-agarose
Summary
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is primarily present within the cell as a homotetramer, comprised of approximately 37 kDa subunits. 2011; Li et al, 2014), and methylation (Forcina et al, 1971) Many of these modifications are linked to the diverse functions of GAPDH, including roles in membrane trafficking, apoptosis, and autophagy (reviewed in Sirover, 2011; Tristan et al, 2011). These roles are in addition to GAPDH’s canonical role within the glycolytic and gluconeogenic pathways for which it is most well known. Such is the case with thirteen-lined ground squirrels, which typically shift to non-carbohydrate fuels during winter hibernation (South & House, 1967; Tashima, Adelstein & Lyman, 1970)
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