Abstract
Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. This reaction has been studied because of its possible role in the in vivo synthesis of psychoactive compounds or neurotoxins and has been characterized biochemically in preparations of rabbit lung. Therefore, we set out to purify rabbit lung INMT, to clone and express its cDNA, and to clone and structurally characterize its gene as steps toward understanding the function and regulation of this enzyme. Rabbit lung INMT was purified and partial amino acid sequence was obtained. A polymerase chain reaction-based approach was then used to clone a rabbit lung INMT cDNA with a 792-base pair open reading frame that encoded a 263-amino acid protein with a predicted molecular mass of 29 kDa. When the cDNA was expressed in COS-1 cells, the encoded protein catalyzed the methylation of tryptamine and structurally related compounds, and was inhibited by two products of the reaction, S-adenosyl-L-homocysteine (AdoHcy) and N,N-dimethyltryptamine, as well as antimigraine drugs that are structurally related to N,N-dimethyltryptamine. Northern blot analysis demonstrated the presence 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 the rabbit INMT gene represent important steps toward determination of the function and regulation of this mammalian methyltransferase enzyme.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF077826 for rabbit lung indolethylamine N-MT (INMT) cDNA and AF077827 and AF077828 for rabbit INMT genes
A series of enzymes capable of methylating tryptamine have been characterized biochemically (2, 8 –12), and at least two tryptamine N-MT isoforms have been described in both rabbit lung [9] and rabbit liver [10, 12]. These activities have been referred to by a variety of names including rabbit lung MT [2], nonspecific MT [2], aromatic alkylamine N-MT [13], indolamine N-MT [9, 14], arylamine N-MT [10], and amine N-MT [12], we will refer to the enzyme that we have studied as indolethylamine N-MT (INMT) [11]
Rabbit Lung INMT cDNA Cloning—The rabbit lung INMT cDNA cloning strategy began with purification of the rabbit lung enzyme
Summary
Materials—[14C-CH3]AdoMet (60 mCi/mmol) and [3H-CH3]AdoMet (56.1 Ci/mmol) were obtained from NEN Life Science Products Inc. (Boston, MA). [␣-32P]dCTP (3000 Ci/mmol) was purchased from Amermethyltransferase; PNMT, phenylethanolamine N-methyltransferase; PCR, polymerase chain reaction; bp, base pair(s); RACE, rapid amplification of cDNA ends; UTR, untranslated region; kb, kilobase pair(s). [␣-32P]dCTP (3000 Ci/mmol) was purchased from Amermethyltransferase; PNMT, phenylethanolamine N-methyltransferase; PCR, polymerase chain reaction; bp, base pair(s); RACE, rapid amplification of cDNA ends; UTR, untranslated region; kb, kilobase pair(s). Materials—[14C-CH3]AdoMet (60 mCi/mmol) and [3H-CH3]AdoMet (56.1 Ci/mmol) were obtained from NEN Life Science Products Inc.
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