Cloning, sequencing, and tissue-dependent expression of flavin-containing monooxygenase (FMO) 1 and FMO3 in the dog.
The expression of flavin-containing monooxygenases (FMOs) in dog liver microsomes was suggested by a high methimazole S-oxidase activity. When the reaction was catalyzed by dog liver microsomes, apparent V(max) and K(m) values were 6.3 nmol/min/mg and 14 microM, 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.
Lattard
Virginie
V
Unité de Toxicologie et de Métabolisme Comparés des Xénobiotiques, Unité Mixte Recherche Institut National de la Recherche Agronomique et Direction Générale de l'Enseignement et de la Recherche, Ecole Nationale Vétérinaire de Lyon, Marcy l'étoile, France.
Longin-Sauvageon
Christiane
C
Lachuer
Joel
J
Delatour
Paul
P
Benoit
Etienne
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000302:isolation & purification; D004285:Dogs; D005415:Flavins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010105:Oxygenases / Q000096:biosynthesis* / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D051381:Rats; D017422:Sequence Analysis, DNA; D013552:Swine
D018076:DNA, Complementary; D005415:Flavins; D007527:Isoenzymes; D012333:RNA, Messenger; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)