Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and other pyridines. Human liver NNMT activity has a bimodal frequency distribution, an observation which raises the possibility that this enzyme activity might be regulated by a genetic polymorphism, a polymorphism that could have functional implications for individual differences in drug and xenobiotic toxicity. As a first step toward testing that hypothesis, we set out to clone and express a cDNA for human liver NNMT. Human liver NNMT was partially purified, photoaffinity-labeled, subjected to limited proteolysis, and partial amino acid sequence information was obtained. The polymerase chain reaction was then used to amplify a 550-nucleotide sequence with human liver cDNA as template and primers designed on the basis of the NNMT amino acid sequence. The 5'- and 3'-ends of a human liver NNMT cDNA were obtained by use of the rapid amplification of cDNA ends. The combined use of these approaches resulted in the isolation of a human liver NNMT cDNA that was 969 nucleotides in length, with a 792-nucleotide open reading frame that encoded a 264-amino acid protein with a calculated molecular mass of 29.6 kDa. The human liver NNMT cDNA was transcribed in vitro and translated with a reticulocyte lysate system to yield a protein with a molecular mass of approximately 29 kDa that comigrated during SDS-polyacrylamide gel electrophoresis with photoaffinity-labeled human liver NNMT. The NNMT cDNA was also subcloned into the eukaryotic expression vector p91023(B). COS-1 cells transfected with this construct expressed a high level of NNMT enzymatic activity, and the biochemical properties of this activity were similar to those of human liver NNMT. Human liver NNMT and transfected COS-1 cell NNMT had apparent K(m) values for the two cosubstrates for the reaction, nicotinamide and S-adenosyl-L-methionine, of 0.43 and 0.38 mM and of 1.8 and 2.2 μM, respectively. IC50 values for the inhibition of NNMT by N1- methylnicotinamide were 60 and 30 μM for human liver and COS-1 cell- expressed NNMT, respectively. Cloning of a cDNA for human liver NNMT will help make it possible to test the hypothesis that inheritance may play a role in the regulation of individual differences in human liver NNMT activity.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology