A highly efficient procedure for purifying the ribosome-activating proteins α- and β-momorcharins from Momordica charantia seeds, N-terminal sequence comparison and establishment of their N-glycosidase activity

A new purification scheme, involving two successive ion exchange chromatographic steps on DEAE-cellulose and Mono-S FPLC, was developed for the isolation of the ribosome-inactivating proteins, α- and β-momorcharins, from the Chinese herb Kuquazi (seeds of Momordica charantia). This simple and rapid procedure yielded 3.1 and 1.7 mg of α- and β-momorcharins, respectively, from 2.5 g of decorticated seeds in only two days. The N-terminal amino acid sequence of β-momorcharin was found to be DVNFDLSTATAKTYTKFIED. It differed from that of α-momorcharin (DVSFRLSGADPRSYGMFIKD) in 10 out of the 20 positions investigated. Like other ribosome-inactivating proteins, the purified momorcharins showed specific N-glycosidase activity at nanomolar concentrations, when rRNA from rabbit reticulocyte lysate was used as substrate. The N-glycosidase activity of both momorcharins was optimal at pH 7, not inhibited by K⁺ and not appreciably affected by NH₄⁺. The activity of α-momorcharin was not drastically altered by Mn²⁺ but (1-10 mM) Mn²⁺ inhibited the activity of β-momorcharin by about 40%.