Abstract
The expression of flavin-containing monooxygenases (FMOs) in dog liver microsomes was suggested by a high methimazoleS-oxidase activity. When the reaction was catalyzed by dog liver microsomes, apparent Vmaxand Km values were 6.3 nmol/min/mg and 14 μM, respectively. This reaction was highly inhibited (73%) in the presence of imipramine, but it was also weakly affected by trimethylamine, suggesting the involvement of different isoforms. The sequences of dog FMO1 and FMO3 were obtained by reverse transcription-polymerase chain reaction and 5′/3′ terminal extension. The cDNAs of dog FMO1 and dog FMO3 encode proteins of 532 amino acids, which contain the NADPH- and FAD-binding sites. The dog FMO1 amino acid sequence is 88, 86, and 89% identical to sequences of human, rabbit, and pig FMO1, respectively. The dog FMO3 amino acid sequence is 83, 84, and 82% identical to sequences of human, rabbit, and rat FMO3, respectively. Dog FMO1 and dog FMO3 exhibited only 56% identities. The FMO1 and FMO3 recombinant proteins and the FMO1 and FMO3 microsomal proteins migrated with the same mobility (56 kDa), as determined in SDS-polyacrylamide gel electrophoresis and immunoblotting. By Western blotting, dog FMO1 and dog FMO3 were detected in microsomes from liver and lung but not in kidney microsomes. By Northern blotting, the probe for FMO1 specifically hybridized a 2.6-kilobase ( kb ) transcript in liver and lung samples only. The probe for FMO3 hybridized two transcripts of approximately 3 and 4.2 kb in the liver and lung samples.
Footnotes
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This study was funded in part by a grant from Direction Générale de l'Enseignement et de la Recherche (DGER) Formation par la Recherche, Ministère de l'Agriculture, France. The nucleotide sequences reported in this article have been submitted to the GenBank under the provided accession number AF384053 for dFMO1 and AF384054 for dFMO3.
- Abbreviations used are::
- FMO
- flavin-containing monooxygenase
- ABT-418
- (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole
- L-775,606
- 3-(3-{4-[2-(3-fluorophenyl)ethyl]piperazin-1-yl}propyl)-5-[1,2,4]triazol-4-yl-1H-indol
- RT-PCR
- reverse transcription-polymerase chain reaction
- MMLV-RT
- Moloney murine leukemia virus-reverse transcriptase
- bp
- base pair
- SSC
- standard sodium citrate
- PAGE
- polyacrylamide gel electrophoresis
- kb
- kilobases
- Received July 12, 2001.
- Accepted October 25, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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