Glutamate dehydrogenase from Escherichia coli: purification and properties.
Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase [deaminating], EC 1.4.1.4) has been purified from Escherichia coli B/r. The purity of the enzyme preparation has been established by polyacrylamide gel electrophoresis, ultracentrifugation, and gel filtration. A molecular weight of 300,000 +/- 20,000 has been calculated for the enzyme from sedimentation equilibrium measurements. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium measurements in guanidine hydrochloride have revealed that glutamate dehydrogenase consists of polypeptide chains with the identical molecular weight of 50,000 +/- 5,000. The results of molecular weight determination lead us to propose that glutamate dehydrogenase is a hexamer of subunits with identical molecular weight. We also have studied the stability and kinetics of purified glutamate dehydrogenase. The enzyme remains active when heat treated or when left at room temperature for several months but is inactivated by freezing. The Michaelis constants of glutamate dehydrogenase are 1,100,640, and 40 muM for ammonia, 2-oxoglutarate, and reduced nicotinamide adenine dinucleotide phosphate, respectively.
D000641:Ammonia / Q000378:metabolism; D000645:Ammonium Sulfate; D002474:Cell-Free System; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D005970:Glutamate Synthase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006358:Hot Temperature; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010455:Peptides / Q000032:analysis
D005971:Glutamates; D007656:Ketoglutaric Acids; D010455:Peptides; D005973:Glutamine; D009249:NADP; D000641:Ammonia; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D000645:Ammonium Sulfate