title: Purification, properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B.
abstract: Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD+-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of greater than 1,092 mumol x min-1 x mg protein-1. The enzyme is a hexamer of a polypeptide of Mr = 42,500, and the native molecular weight is 250,800. The apparent Km values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD+ in the deamination reaction, and 7.2 mM for alpha-ketoglutarate, 243 mM for NH4+ and 0.028 mM for NADH in the reverse reaction. NADP+ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate alpha-ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.
authors.affiliation: Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706.
authors.forename: B A
authors.initials: BA
authors.lastname: Hammer
authors.forename: E A
authors.initials: EA
authors.lastname: Johnson
mesh_terms: D003014:Clostridium botulinum / Q000201:enzymology*; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005971:Glutamates; D009243:NAD; D005969:Glutamate Dehydrogenase