Abstract
The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library. Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration. The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively. The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Chromatography, Ion Exchange
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Cloning, Molecular
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DNA Primers
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Diethyl Pyrocarbonate / pharmacology
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / genetics
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Gene Expression / genetics
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Humans
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Imidoesters / pharmacology
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Iodoacetamide / pharmacology
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Kinetics
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Methyl Methanesulfonate / analogs & derivatives
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Methyl Methanesulfonate / pharmacology
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Molecular Sequence Data
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Phenylethanolamine N-Methyltransferase / chemistry
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Phenylethanolamine N-Methyltransferase / genetics*
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Phenylethanolamine N-Methyltransferase / isolation & purification
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Phenylglyoxal / pharmacology
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Plasmids / genetics
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Recombinant Proteins / genetics*
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Recombinant Proteins / isolation & purification
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S-Adenosylmethionine / metabolism
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Tetranitromethane / pharmacology
Substances
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DNA Primers
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Imidoesters
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Recombinant Proteins
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methyl methanethiosulfonate
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S-Adenosylmethionine
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Methyl Methanesulfonate
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methyl acetimidate
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Phenylethanolamine N-Methyltransferase
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Tetranitromethane
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Diethyl Pyrocarbonate
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Phenylglyoxal
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Iodoacetamide