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 degrees C body temperature) and hibernating (torpid, 5 degrees 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 Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees 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.
Thatcher
B J
BJ
Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, ON, Canada.
Storey
K B
KB
D000818:Animals; D001831:Body Temperature; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D006605:Hibernation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D012589:Sciuridae / Q000502:physiology*
D007527:Isoenzymes; D005969:Glutamate Dehydrogenase