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 K(m) 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. © 1993 Academic Press, Inc.