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.
Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, ON, Canada.
B J
BJ
Thatcher
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Storey
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