Enzymatic Synthesis of Sinapine from 1-O-Sinapoyl-β-ᴅ-glucose and Choline by a Cell-Free System from Developing Seeds of Red Radish (Raphanus sativus L. var. sativus)

Abstract

A cell-free system from developing seeds of red radish (Raphanus sativus L. var. sativus) catalyzed the transfer of the sinapoyl moiety of 1-O-sinapoyl-β-ᴅ-glucose to choline to form O-sinapoylcholine (sinapine). A wide range of proportionality with respect to incubation time and protein concentration was found in the determination of this enzymatic activity using a high performance liquid chromatographic (HPLC) method. The transacylase activity showed an absolute acceptor specificity towards choline and a high hydroxycinnamoyl donor specificity with 1-sinapoylglucose. Maxim|al rate of sinapine formation was found to be around pH 7.0 in 0.08 M potassium phosphate buffer. The apparent K_m values were 0.30 mм for 1-sinapoylglucose and 7.64 mм for choline. Change in extractable transacylase activities from different stages of seed development correlated well with the in vivo accumulation kinetics of sinapine. Maximal activity around 36 pkat seed^-1 and 129μkat kg^-1 protein was extracted from dark-green immature seeds, which are in the process of rapid sinapine accumulation. About 20% of this activity still has been found in dark-brown mature seeds. This enzymatic acitivity refers to an enzyme which can be classified as 1-sinapoylglucose: choline sinapoyltransferase (SCT).