Glutamate dehydrogenase from Bacillus subtilis PCI 219. I. Purification and properties.
Bacillus subtilis PCI 219 has a single glutamate dehydrogenase (GDH) [EC 1.4.1.3] with dual coenzyme specificity [for NAD(H) and NADP(H)]. The enzyme was purified 800-fold from crude extracts of B. subtilis from the post-exponential phase of growth and showed one significant protein band on gel electrophoresis. This band was determined, by activity staining, to have all the GDH nucleotide specificities. Its molecular weight was estimated to be 250,000+/-20,000 by gel filtration, and 270,000+/-30,000 by zone centrifugation in a sucrose density gradient. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that GDH has a subunit size of about 57,000. The pI of GDH was found to bepH 3.7 by isoelectric focusing. GDH exhibited nonlinear kinetics in the reduction of NAD+, and in the reverse direction, the substrate, NH4+, was strongly inhibitory at high concentrations. Purine nucleotides did not affect the activity. The oxidative demination of glutamate was significantly inhibited by the metabolites oxaloacetate and citrate, which acted as allosteric effectors of this enzyme,inhibiting the reaction in one direction. The pH optimum of each of the activities of GDH and the stability of GDH are also reported.
D001412:Bacillus subtilis / Q000201:enzymology*; D002951:Citrates / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010071:Oxaloacetates / Q000494:pharmacology; D011685:Purine Nucleotides / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
D002951:Citrates; D007656:Ketoglutaric Acids; D010071:Oxaloacetates; D011685:Purine Nucleotides; D000644:Quaternary Ammonium Compounds; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase