A HIGHLY EFFICIENT PROCEDURE FOR PURIFYING THE RIBOSOME-INACTIVATING PROTEINS ALPHA- AND BETA-MOMORCHARINS FROM MOMORDICA CHARANTIA SEEDS, N-TERMINAL SEQUENCE COMPARISON AND ESTABLISHMENT OF THEIR N-GLYCOSIDASE ACTIVITY

摘要

A new purification scheme, involving two successive ion exchange chromatographic steps on DEAF-cellulose and Mono-S FPLC, was developed for the isolation of the ribosome-inactivating proteins, alpha- and beta-momorcharins, from the Chinese herb Kuquazi (seeds of Momordica charantia). This simple and rapid procedure yielded 3.1 and 1.7 mg of alpha- and beta-momorcharins, respectively, from 2.5 g of decorticated seeds in only two days. The N-terminal amino acid sequence of beta-momorcharin was found to be DVNFDLSTATAKTYTKFIED. It differed from that of alpha-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 pH7, not inhibited by K+ and not appreciably affected by NH4+. The activity of alpha-momorcharin was not drastically altered by Mn2+ but (1-10mM) Mn2+ inhibited the activity of beta-momorcharin by about 40%. [References: 25]