Molecular cloning of cDNA and chromosomal assignment of the gene for human phenylethanolamine N-methyltransferase, the enzyme for epinephrine biosynthesis.
Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the synthesis of epinephrine from norepinephrine, the last step of catecholamine biosynthesis. To isolate a cDNA clone for human PNMT, we first isolated a cDNA clone for bovine adrenal medulla PNMT using mixed oligodeoxyribonucleotide probes whose synthesis was based on the partial amino acid sequence of tryptic peptides from the bovine enzyme. By screening a bovine adrenal medulla cDNA library, a cDNA clone with an insert of about 200 base pairs (bp) was isolated. This clone consisted of 84 bp of carboxyl-terminal coding region, which contained amino acid sequences corresponding to two tryptic peptides, and about 100 bp of 3'-untranslated region. Using this cDNA fragment as the probe, we screened a human pheochromocytoma cDNA library and isolated a cDNA clone with an insert of about 1.0 kilobase pairs, which contained the complete coding region of the enzyme. Northern blot analysis of human pheochromocytoma poly(A)+ RNA using this cDNA insert as the probe showed a single RNA species of about 1,000 nucleotides, suggesting that this clone is a full-length cDNA. Determination of the nucleotide sequence revealed that human PNMT consists of 282-amino acid residues with a predicted molecular weight of 30,853, including initial methionine. The amino acid sequence of the human PNMT was highly homologous (88%) to that of the bovine enzyme. Chromosomal assignment of the gene for human PNMT was carried out using mouse-human somatic cell hybrids. The PNMT gene was assigned to chromosome 17.
Kaneda
N
N
Department of Biochemistry, Nagoya University School of Medicine, Japan.
Ichinose
H
H
Kobayashi
K
K
Oka
K
K
Kishi
F
F
Nakazawa
A
A
Kurosawa
Y
Y
Fujita
K
K
Nagatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004837:Epinephrine / Q000096:biosynthesis*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics*
D004247:DNA; D010625:Phenylethanolamine N-Methyltransferase; D004837:Epinephrine