Formation of hydrogen peroxide and N-hydroxylated amines catalyzed by pulmonary flavin-containing monooxygenases in the presence of primary alkylamines

In atypical reaction, incubation of purified rabbit pulmonary flavin-containing monooxygenase with certain primary alkylamines results in the oxidation of NADPH and the formation of hydrogen peroxide. In addition, significant amounts of N-hydroxylated primary amine are also generated, as determined by colorimetric assay and GC/MS analysis of n-octylamine metabolites. Similar reactions appear to be catalyzed by the mouse pulmonary enzyme. In contrast, incubation of primary alkylamines with hepatic flavin-containing monooxygenases from rabbit, mouse, or pig does not result in NADPH oxidation or metabolism. Another effect of primary alkylamines is marked activation of the mouse pulmonary and pig hepatic flavin-containing monooxygenases with some substrates. The structural requirements for primary alkylamines to elicit NADPH oxidation by the rabbit pulmonary enzyme or to activate the mouse pulmonary and pig hepatic enzymes are identical. This indicates that different flavin-containing monooxygenases probably have a conserved alkylamine-binding site of defined specificity. In the case of the rabbit pulmonary enzyme, this binding may occur very close to or at the catalytic site resulting in some N-hydroxylation of the alkylamine.     

The nutritional value of seven d-amino acids and α-ketoacids for Argyrotaenia velutinana,Heliothis zea,and Phormia regina

The d-isomers of methionine, phenylalanine, and histidine were the only d-isomers which replaced their l-enantiomeric forms to some extent in the diets for Argyrotaenia velutinana and Heliothis zea. In addition to these 3 d-isomers Phormia regina could utilize d-leucine and d-tryptophan. None of the α-ketoacids could be utilized by A. velutinana and H. zea. P. regina could utilize the α-ketoacids of leucine and methionine.     

Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior

Equilibrium titrations in N-methylformamide (NMF) of G-25 gel filtered (ox)-state FeMo cofactor [FeMoco(ox)] from Azotobacter vinelandii nitrogenase were carried out using sodium ethanethiolate and followed using UV/Vis absorption spectroscopy. For Fe-Moco(ox), a non-linear least squares (NLLSQ) fit to the data indicated a strong equilibrium thiolate-binding step with Keq = 1.3+/-0.2x10(6) M(-1). With 245 molar excess imidazole, cooperative binding of three ethanethiolates was observed. The best NLLSQ fit gave Keq=2.0+/-0.1x10(5) M(-2) and a Hill coefficient n=2.0+/-0.3. A Scatchard plot of these data was concave upward, indicating positive cooperativity. The fit to previously published data involving benzenethiol titration of the one-electron reduced (semi-reduced) cofactor, FeMoco(sr), as followed by EPR required a model that included both a sub-stoichiometric ratio of thiol to FeMoco(sr) and about five cooperative ligand binding sites. These constraints were met by modeling FeMoco(sr) as an aggregate, with fewer thiol binding sites than FeMoco(sr) units. The best fit model was that of FeMoco(sr) as a dodecamer with five cooperative benzenethiol binding sites, yielding a thiol binding constant of 3.32+/-0.09x10(4) M(-4.8) and a Hill coefficient n=4.8+/-0.6. The results of all the other published ligand titrations of FeMoco(sr) were similarly analyzed successfully in terms of equilibrium models that include both cooperative ligand binding and dimer-level aggregation. A possible structural model for FeMoco aggregation in NMF solution is proposed.     

Discrimination by heat and proteinase treatments between flocculent phenotypes conferred on Saccharomyces cerevisiae by the genes FLO1 and FLO5

The effects of elevated temperature and of digestion with a variety of proteinases on the flocforming ability of flocculent strains of Saccharomyces cerevisiae, both genetically defined (FLO1 and FLO5) laboratory and genetically undefined brewing strains, have been determined. This has permitted classification of the flocculent phenotypes of these strains according to criteria other than quantitative grading of flocculence. The flocculent phenotypes conferred by both the FLO1 and the FLO5 gene were irreversibly lost upon treatment with pronase, proteinase K, trypsin or 2-mercaptoethanol treatments. However, the floc-forming ability of cells of the FLO1 strain ABXL-1D was destroyed by chymotrypsin digestion and was stable to incubation at 70 degrees C, whereas the floc-forming ability of cells of the FLO5 strain ABXR-11A was resistant to the action of chymotrypsin and was heat labile. Tetrad analysis of a cross of these FLO1 and FLO5 strains indicated that the chymotrypsin and heat sensitivity phenotypes were FLO-gene determined. It appears that expression of the FLO1 and FLO5 genes leads to the production of different and characteristic cell-wall proteins underlying their respective flocculent phenotypes.