Comparative studies on S-adenosyl-L-methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-L-methionine as photoaffinity probe.
Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-L-[methyl-3H]methionine (8-N3-Ado[methyl-3H]Met), the binding sites for S-adenosyl-L-methionine(AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC 2.1.1.23); AdoMet:histone-arginine N-methyltransferase (EC2.1.1.23); and AdoMet:cytochrome c-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.
Syed
S K
SK
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140.
Kim
S
S
Paik
W K
WK
D000345:Affinity Labels / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002918:Chymotrypsin; D008099:Liver / Q000201:enzymology; D008716:Methionine Adenosyltransferase / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011496:Protein Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D014316:Tritium; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D010446:Peptide Fragments; D014316:Tritium; D012436:S-Adenosylmethionine; C037207:8-azido-S-adenosylmethionine; D011496:Protein Methyltransferases; D008716:Methionine Adenosyltransferase; D002918:Chymotrypsin; D014357:Trypsin