Abstract
A full-length dog (beagle) flavin-containing monooxygenase 1 (FMO1) cDNA (dFMO1) was obtained from liver by reverse transcription-polymerase chain reaction. The amino acid sequence of dFMO1 was 89% homologous to human FMO1. Using a baculovirus expression system in Sf-9 insect cells, dFMO1 was expressed to protein levels of 0.4 nmol/mg, as determined by immunoquantitation. The flavin content of the expressed enzyme was consistent with immunodetectable dFMO1 protein levels. Expressed dFMO1 catalyzed NADPH-dependent methyl p-tolyl sulfide oxidation, withKm and Vmaxvalues of 98.6 μM and 63.8 nmol of S-oxide formed/min/mg of protein, respectively. By comparison, human FMO1 showed similar values of 87.1 μM (Km) and 51.0 nmol/min/mg (Vmax). Activity for dFMO1 showed characteristic pH dependence, with a 4.5-fold increase inS-oxidase activity as the incubation pH increased from 7.6 to 9.0. Human FMO1 also showed an increase in reaction rate with pH but a somewhat lower optimum of 8.0 to 8.4. dFMO1 also catalyzed imipramine N-oxidation, with aKm of 4.7 μM and aVmax of 82.1 nmol/min/mg of protein. This enzyme displayed other characteristics of FMO enzymes, with rapid depletion of enzyme activity upon heating in the absence of NADPH. Protein levels of 74 pmol of dFMO1/mg of microsomal protein were determined for a pooled liver microsome sample, suggesting that this enzyme is a major canine hepatic monooxygenase. In conclusion, the expression and characterization of catalytically active dFMO1 will allow the role of this enzyme in the metabolism of xenobiotics to be determined.
Footnotes
- Abbreviations:
- FMO
- flavin containing monooxygenase
- P450
- cytochrome P450
- dFMO1
- dog FMO1
- hFMO1
- human FMO1
- MPT
- methylp-tolyl
- MPTS
- methyl p-tolyl sulfide
- Sf-9
- Spodoptera frugiperda
- tricine
- N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
- Received October 1, 2002.
- Accepted November 8, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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