Histamine N-methyltransferase from rat kidney. Cloning, nucleotide sequence, and expression in Escherichia coli cells. |
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Authors: | M Takemura T Tanaka Y Taguchi I Imamura H Mizuguchi M Kuroda H Fukui A Yamatodani H Wada |
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Institution: | Department of Pharmacology II, Osaka University Faculty of Medicine, Suita, Japan. |
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Abstract: | Complementary DNA clones encoding rat kidney histamine N-methyltransferase have been isolated using synthetic oligonucleotide probes based on partial amino acid sequences of tryptic peptides of the purified enzyme. The 1.3-kilobase cDNA consisted of a 5'-noncoding region of 8 nucleotides, a coding region of 885 nucleotides, and a 3'-noncoding region of 369 nucleotides. The encoded protein of 295 amino acid residues had a calculated molecular weight of 33,940.2. After introduction of a prokaryotic expression vector containing the isolated cDNA, Escherichia coli cells expressed histamine N-methyltransferase activity. The enzyme expressed in these cells was isolated and purified as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whose mobility was identical to the natural enzyme purified from rat kidney. The recombinant enzyme had Vmax and Km values for both histamine and S-adenosylmethionine identical to those of the natural enzyme. All of the inhibitors of the natural enzyme tested showed similar Ki values on both recombinant and natural enzyme. |
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