title: Subcellular localization and properties of partially purified dimethylamine and trimethylamine mono-oxygenase activities in Candida utilis.
abstract: By techniques involving differential centrifugation and specific precipitation with CaCl2, it was shown that dimethylamine and trimethylamine mono-oxygenase activities co-sediment with NADPH-cytochrome c reductase activity in sphaeroplast lysates of Candida utilis grown on trimethylamine as sole nitrogen source. Since the active fraction also contained low levels of cytochromes P-450 and P-420, it was concluded that the two amine mono-oxygenases are located in the smooth endoplasmic reticulum and thus end up in the microsomal fraction on cell fractionation. Ten to twenty-fold enrichment of mono-oxygenase specific activity could be achieved by separation of activity from soluble protein by centrifugation or gel filtration. Cell-free extracts prepared in the absence of FAD showed only very low mono-oxygenase activity for either substrate. Some activity could be restored by addition of flavin nucleotides: there was a fivefold stimulation by FAD and a fourfold stimulation by FMN. All trimethylamine mono-oxygenase activity was lost when a partially purified preparation containing both activities was incubated for more than 24 h at 0 degrees C, suggesting that separate enzymes are responsible for the oxidation of secondary and tertiary amines. The enzyme preparation oxidized a wide range of secondary alkylamines up to dibutylamine and tertiary alkylamines up to tributylamine. Primary amines, choline, di- and triethanolamine, spermine, spermidine and substituted anilines were not oxidized. NADH had a lower apparent Km value and higher Vmax value than NADPH. Secondary and tertiary alkylamines containing more than one kind of alkyl group gave more than one kind of aldehyde on oxidation. Stoicheiometry determinations showed a consumption of 1 mol NAD(P)H and 1 mol O2 per mol aldehyde formed. Carbon monoxide, cyanide, proadifen hydrochloride (SKF 525-A), mercurials and mercaptoethanol all inhibited both activities.
mesh_terms: D002175:Candida / Q000201:enzymology*; D002498:Centrifugation; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D010105:Oxygenases; C022626:secondary amine monooxygenase; C043996:tertiary amine monooxygenase