Glutamate dehydrogenase from Escherichia coli: purification and properties

Abstract

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase [deaminating], EC 1.4.1.4) has been purified from E. 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 to the proposal that glutamate dehydrogenase is a hexamer of subunits with identical molecular weight. The stability and kinetics of purified glutamate dehydrogenase were also studied. The enzyme remains active when heat treated or when left at room temperature for several months but it is inactivated by freezing. The Michaelis constants of glutamate dehydrogenase are 1,100, 640, and 40 μM for ammonia, 2 oxoglutarate, and reduced nicotinamide adenine dinucleotide phosphate, respectively.