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

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37%C body temperature) and hibernating (torpid, 5%C body temper ature) 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 33 5 +/- 5 kDa for euthermic and 320 +/- 5 kDa for hibernator GDH. Euthermic a nd hibernator GDH differed substantially in apparent K-m values for glutama te, NH4+, and alpha-ketoglutarate, as well as in K-a and IC50 values for nu cleotide and ion activators and inhibitors. Kinetic properties of each enzy me were differentially affected by assay temperature (37 versus 5%C). For e xample, the K-m for alpha-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 alpha-ketoglutarate at 5%C (K-m was 0.98 +/- 0.08 mM at 37%C and 0.43 +/- 0.02 mM at 5%C). Temperature effects on K-a ADP va lues of the enzymes followed a similar pattern; GTP inhibition was stronges t 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 prev ailing body temperature.