Rabbit lung indolethylamine N-methyltransferase - cDNA and gene cloning and characterization

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of t ryptamine and structurally related compounds. This-reaction has been studie d because of its possible role in the in vivo synthesis of psychoactive com pounds or neurotoxins and has been characterized biochemically in preparati ons of rabbit lung. Therefore, we set out to purify rabbit lung INMT, to cl one and express its cDNA, and to clone and structurally characterize its ge ne as steps toward understanding the function and regulation of this enzyme . Rabbit lung INMT was purified and partial amino acid sequence was obtaine d, A polymerase chain reaction-based approach was then used to clone a rabb it lung INMT cDNA with a 792-base pair open reading frame that encoded a 26 3-amino acid protein with a predicted molecular mass of 29 kDa. When the cD NA was expressed in COS-1 cells, the encoded protein catalyzed the methylat ion of tryptamine and structurally related compounds, and was inhibited by two products of the reaction, S-adenosyl-L-homocysteine (AdoHcy) and N,N-di methyltryptamine, as well as antimigraine drugs that are structurally relat ed to N,N-dimethyltryptamine. Northern blot analysis demonstrated the prese nce of 2.0-kilobase mRNA species in rabbit lung, liver and, at lower levels , in brain, The cDNA was then used to clone the rabbit INMT gene. That gene had three exons and was structurally similar to the genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. Cloning and expression of a rabbit lung INMT cDNA and cloning of t he rabbit INMT gene represent important steps toward determination of the f unction and regulation of this mammalian methyltransferase enzyme.