NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.
GC DETERMINATION OF THIOFANOX RESIDUES tion curves indicate the presence of carboxylate ions that could be explained by the presence of Na salts of some organic acids as impurities.Elementary analyses of the model substance gave the simplest empirical formula of CigH260nN, whereas the empirical formula of the isolate from barley roasted at 250°f or 40 min was found to be CigHgvOnN. Elementary analysis of both the model substance and the isolate from roasted barley indicated that they are of similar composition.The manner by which nitrogen is chemically bound in the brown substance, produced by the process of nonenzymatic browning after the temperature treatment, is not understood. The concentration of the brown substance in roasted barley was unexpectedly high. However, it can be explained by the fact that nonenzymatic browning occurs in two ways. The first occurs from the reaction between amino acid and reducing sugar, and the second occurs from caramelization, which can, but need not, include nitrogen.Isolation of the brown pigments from roasted barley by means of ion-exchange resin Permutit ES confirms that the substance is of an ionic nature and has a negative charge. According to the results obtained from uv and ir spectra (Figures 1 and 2) and from elementary analysis it was concluded that the isolate of the brown substance from roasted barley was of similar composition to the model substance.
ACKNOWLEDGMENTThe able technological assays of M. Glogovcan are grate-fully acknowledged.
N-dealkylated metabolites, deethylatrazine and deisopropylatrazine, are further hydrolyzed to the corresponding hydroxy analogues.
ACKNOWLEDGMENTThe skilled technical assistance of N.
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