Glutamic dehydrogenase from rat heart mitochondria. I. Purification and physical properties including molecular weight determination.
Glutamic dehydrogenase extracted with tris buffer from fresh freeze-thawed rat heart mitochondria was purified by ammonium sulphate fractionation, affinity chromatography on GTP agarose, hydroxyapatite chromatography and concentration using a molecular sieve. The final specific activity is 80 units/mg protein. Thin gel SDS electrophoresis of the purified enzyme preparation after reduction with dithiothreitol shows a major band with a molecular weight of 38 000 Daltons. Two minor bands are also present. Sucrose density gradient centrifugation reveals a molecular weight of 230 000 Daltons for unreduced mitochondrial GDH activity. By gel filtration rat heart mitochondrial glutamic dehydrogenase has a major peak at 230 000 Daltons, a minor peak at 300 000 Daltons and some larger molecular weight species. Rat liver mitochondrial glutamic dehydrogenase has a minor peak at 230 000, a major peak at 300 000 and some larger molecular weight species. The rat liver mitochondrial glutamic dehydrogenase predominance at 300 000 is unchanged by incubation, extraction and purification with rat heart mitochondria. The purified GDH is stable frozen at -10 degrees C in tris-HCl buffer with EDTA. It loses activity at 4 degrees C especially when stored in 0.2 M phosphate buffer. It also loses activity when dialyzed for 24 h. This loss of activity is not completely prevented by adding nucleotides to the buffer (AMP or ADP) but is decreased by their presence.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000032:analysis / Q000302:isolation & purification*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D051381:Rats
D005969:Glutamate Dehydrogenase