title: Covalently bound FAD in d-6-hydroxynicotine oxidase. Immunological studies of D- and L-6-hydroxynicotine oxidase: evidence for a D-enzyme precursor.
abstract: Antersera prepared against both enantiozymes, D- and L-6-hydroxynicotine oxidase, formed precipitins in double diffusion tests with their respective antigens only. A mixture of the two antisera caused spur formation of the two precipitin lines obtained with the pure enzymes. Antiserum to L-apoprotein reacted with native L-enzyme and L-apoprotein but not with the D-sspecific enzyme. D-6-hydroxynicotine oxidase activity was inhibited by the anti-D-antiserum, leaving the L-enzyme fully active, while anti-L-antiserum inhibited the L- but not the D-specific activity. The delayed induction of D-6-hydroxynicotine oxidase as compared to the other activities of the nicotine-degrading sequence and the differential immunochemical behavior of the enantiozymes allowed the search for a D-enzyme precursor. In cells harvested 3 hours after the addition of DL-nicotine, the L-enzyme activity was present, whereas no D-enzyme activity could be detected. However, an extract of these cells did form an immunoprecipitin line with anti-D-antiserum. L-6-Hydroxynicotine oxidase, but no D-6-hydroxynicotine oxidase activity, could also be induced in Arthrobacter oxidans grown in a medium with a high glucose content and DL-nicotine as the sole nitrogen source. An extract of these L-induced cells produced the specific immunoprecipitation with anti-D-antiserum. A pulse-chase experiment with cells grown first on glucose and DL-nicotine in the presence of [14C]leucine and then in an unlabeled medium which induces D-6-hydroxynicotine oxidase activity resulted in a radioactive D-enzyme-immunoprecipitin line. From these experiments it is concluded that a precursor of the active D-enzyme is induced simultaneously with the other nicotine-degrading enzymes.
mesh_terms: D001173:Arthrobacter / Q000187:drug effects / Q000201:enzymology; D004790:Enzyme Induction; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis*; D005947:Glucose / Q000494:pharmacology; D005779:Immunodiffusion; D007700:Kinetics; D009538:Nicotine / Q000031:analogs & derivatives; D010088:Oxidoreductases / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D013237:Stereoisomerism
chemical_list: D005182:Flavin-Adenine Dinucleotide; D009538:Nicotine; D010088:Oxidoreductases; D005947:Glucose