Purification and partial amino acid sequence of bovine adrenal phenylethanolamine N-methyltransferase: a comparison of nucleic acid and protein sequence data.
Recently, we have reported the isolation and characterization of a putative genomic DNA clone encoding bovine adrenal phenylethanolamine N-methyltransferase (PNMT) (Batter et al., 1988). However, the lack of primary amino-acid sequence data for this enzyme precluded a definitive proof of the authenticity of this clone. To establish identity, the amino acid sequence of several peptides generated by chemical and enzymatic hydrolysis of purified PNMT was compared to that predicted from the nucleotide sequence of the exons of the putative PNMT gene. Bovine adrenomedullary PNMT was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Treatment with 70% formic acid cleaved the protein at a single Asp-Pro bond near the N-terminus. The purified protein was also cleaved at a single methionine residue near the C-terminus by treatment with cyanogen bromide. N-terminal amino acid sequence analysis identified 8 and 10 amino acid residues, respectively, following each of the scissile peptide bonds. Four tryptic peptides, generated by complete enzymatic digestion, were isolated by reverse-phase HPLC and subjected to sequence analysis. Combined, the amino acid sequences of these six peptides represent 20% of the PNMT protein. These amino acid sequences matched exactly the sequences predicted from the exons of the putative PNMT genomic clone.
Weisberg
E P
EP
Department of Psychiatry, Western Psychiatric Institute and Clinic, Pittsburgh, Pennsylvania 15213.
Batter
D K
DK
Brown
W E
WE
Kaplan
B B
BB
D000313:Adrenal Medulla / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics* / Q000302:isolation & purification
D010625:Phenylethanolamine N-Methyltransferase