Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: Evidence for two distinct enzyme forms

Abstract

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37°C body temperature) and hibernating (torpid, 5°C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5 ± 2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335 ± 5 kDa for euthermic and 320 ± 5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Kmvalues for glutamate, NH4+, and α-ketoglutarate, as well as in Kaand IC50values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5°C). For example, the Kmfor α-ketoglutarate of euthermic GDH was higher at 5°C (3.66 ± 0.34 mM) than at 37°C (0.10 ± 0.01 mM), whereas hibernator GDH had a higher affinity for α-ketoglutarate at 5°C (Kmwas 0.98 ± 0.08 mM at 37°C and 0.43 ± 0.02 mM at 5°C). Temperature effects on KaADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37°C and weakest with hibernator GDH at 5°C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.Key words: mammalian hibernation, amino acid metabolism, temperature-dependent enzyme kinetics, Spermophilus richardsonii.