HUMAN HISTAMINE N-METHYLTRANSFERASE PHARMACOGENETICS - CLONING AND EXPRESSION OF KIDNEY CDNA

Histamine N-methyltransferase (HNMT) catalyzes the N(tau)-methylation of histamine. The level of HNMT activity in human red blood cells is c ontrolled by a common genetic polymorphism. We set out to clone and ex press a cDNA for HNMT from human tissue as a first step toward a deter mination of the molecular basis for this genetic polymorphism. The clo ning strategy was based on possible sequence homology between rat and human kidney HNMT. Human kidney cDNA libraries were screened with the 885-nucleotide open reading frame of rat kidney HNMT cDNA. A 1.4-kilob ase cDNA clone was isolated that contained two potential translation i nitiation codons, both in the same reading frame. The longest open rea ding frame of the human kidney cDNA clone contained 876 nucleotides an d encoded a protein 292 amino acids in length. The amino acid sequence of this protein was 84% identical to that of rat kidney HNMT. The hum an kidney cDNA clone was transcribed in vitro and translated in a rabb it reticulocyte lystate system to yield a protein with an apparent mol ecular mass of 33 kDa, as estimated by sodium dodecyl sulfate-polyacry lamide gel electrophoresis. The human kidney cDNA was also subcloned i nto the eukaryotic expression vector p91023(B). Partially purified HNM T isolated from the cytosol of COS-1 cells transfected with this expre ssion construct had biochemical properties similar to those of human k idney HNMT. Human renal cortical HNMT, partially purified human renal cortical HNMT, and partially purified transfected COS-1 cell HNMT had K(m) values for histamine and S-adenosyl-L-methionine, the two cosubst rates for the enzyme reaction, of 20, 13, and 14 muM and 2.0, 3.0, and 6.2 muM, respectively. IC50 values for the HNMT inhibitor amodiaquine were 0.50, 0.48, and 0.40 muM, respectively, for enzyme from these sa me three sources. Northern blot analyses performed with poly(A)+ RNA f rom a series of human tissues including kidney demonstrated three tran scripts, approximately 1.3, 3.8, and 4.0 kilobases in length. Cloning of a cDNA for HNMT may now make it possible to determine the molecular basis for the HNMT genetic polymorphism in humans.