Purification and characterization of S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase from suspension cultures of alfalfa (Medicago sativa L.).
Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit Mr 41,000. COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosyl-methionine and S-adenosylhomocysteine. Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates. An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity. Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins. The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.
Edwards
R
R
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402.
Dixon
R A
RA
D000345:Affinity Labels; D002109:Caffeic Acids / Q000378:metabolism; D002478:Cells, Cultured; D002845:Chromatography; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D000455:Medicago sativa / Q000201:enzymology*; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010449:Peptide Mapping; D010777:Photochemistry; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity
D000345:Affinity Labels; D002109:Caffeic Acids; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C019608:caffeate O-methyltransferase; C040048:caffeic acid