Purification and characterization of sinapine synthase from seeds of Brassica napus.
1-O-Sinapoylglucose:choline sinapoyltransferase ("sinapine synthase") has been purified from immature seeds of Brassica napus by sequential hydroxylapatite absorption, ion-exchange chromatography, and gel filtration. The purified enzyme has an apparent molecular weight of 65 kDa on gel filtration and a subunit structure on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 28 kDa. Sinapine synthase has Km values in the high micromolar range for both substrates (1-O-sinapoylglucose and choline chloride) but these values are sensitive to the concentration of the second substrate. The enzyme displays a marked substrate specificity for 1-O-sinapoylglucose among other related glucose esters. No requirements for thiol protectants or divalent cations were found, but sinapine synthase activity is inhibited by Cu2+ and Hg2+ ions. Partial amino acid sequence data have been obtained from a tryptic digest of the 28-kDa polypeptide.
Vogt
T
T
Department of Plant Science, University of British Columbia, Vancouver, Canada.
Aebershold
R
R
Ellis
B
B
D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D001937:Brassica / Q000201:enzymology* / Q000254:growth & development; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012639:Seeds / Q000201:enzymology* / Q000502:physiology
D000596:Amino Acids; D006882:Hydroxyapatites; D010446:Peptide Fragments; D017886:Durapatite; D000217:Acyltransferases; C079635:sinapoylglucose-choline O-sinapoyltransferase