Characterization of liver flavin-containing monooxygenase of the dogfish shark (Squalus acanthias) and partial purification of liver flavin-containing monooxygenase of the silky shark (Carcharhinus falciformis).
Flavin-containing monooxygenase (FMO) activity as N,N-dimethylaniline (DMA) N-oxygenation was characterized in microsomes from the smooth dogfish shark (Squalus acathias). DMA N-oxygenase activity from the liver of the dogfish shark was linear with increasing protein content and over 60 min. The optimal temperature for catalysis was 25 degrees C with a 76 percent reduction in activity when incubated at 15 degrees C and 99 percent loss of activity at 45 degrees C. Optimal pH was approximately 9.6. The maximum velocity for DMA N-oxygenase activity was calculated to be 1.3 nmol min-1 mg-1 with an apparent Michaelis constant of 44 microM. Methimazole oxidase activity was also observed in dogfish liver microsomes which was inhibited by trimethylamine (TMA). Inhibition of DMA N-oxygenase activity by TMA and thiobenzamide was competitive, while inhibition by methimazole was not competitive. Western blot analysis indicated a single liver protein from both Squalus and Carcharhinus of approximately 50 kDa that bound to antibodies raised against FMO 2. An attempt was made to purify FMO as methimazole oxidase from the liver of the silky shark. A single peak of about 10-fold purity was observed following passage through two chromatographic media (CM-Sepharose and HA-Agarose). However, no activity was recoverable after the FMO-containing fractions were applied to a 2'5' ADP-Sepharose column.
Schlenk
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Division of Toxicology, University of Arkansas for Medical Sciences, Little Rock 72205.
Li-Schlenk
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D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D004789:Enzyme Activation / Q000187:drug effects; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008744:Methylamines / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D010105:Oxygenases / Q000032:analysis*; D012754:Sharks
D008744:Methylamines; D005486:Flavin Mononucleotide; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); C023336:trimethylamine