title: Identification of distinct NAD-linked hydrogenase protein species in mutants and nickel-deficient wild-type cells of Alcaligenes eutrophus H16.
abstract: By crossed immunoelectrophoresis with antibodies against the NAD-linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16. Protein 1 (antigen 1) exhibited NAD-reducing activity and was shown to be identical with the native heterotetrameric enzyme. Protein 2 (antigen 2) was catalytically inactive in the antibody-precipitated form and corresponded to the beta subunit (56 kDa) of the holoenzyme. Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH-oxidizing (diaphorase) activity, suggesting that it either consists of the alpha peptide or of the alpha and gamma subunits of the diaphorase dimer. Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency. Low concentrations of the diaphorase-active antigen 3 were also detected under these conditions. Extracts from mutants defective in the catalytic activity of NAD-reducing hydrogenase still contained the four polypeptides. This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits. However, as observed with nickel-deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70-80%) was the beta peptide.
authors.affiliation: Institut für Mikrobiologie, Universität Göttingen, Federal Republic of Germany.
authors.forename: S
authors.initials: S
authors.lastname: Hornhardt
authors.forename: K
authors.initials: K
authors.lastname: Schneider
authors.forename: B
authors.initials: B
authors.lastname: Friedrich
authors.forename: B
authors.initials: B
authors.lastname: Vogt
authors.forename: H G
authors.initials: HG
authors.lastname: Schlegel
mesh_terms: D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007118:Immunoassay; D005779:Immunodiffusion; D007123:Immunoelectrophoresis, Two-Dimensional; D009154:Mutation; D009532:Nickel / Q000494:pharmacology*; D010088:Oxidoreductases / Q000032:analysis* / Q000235:genetics; D010641:Phenotype
chemical_list: D009532:Nickel; D010088:Oxidoreductases; C021376:hydrogen dehydrogenase