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.
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, 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%.
Fong
W P
WP
Department of Biochemistry, Chinese University of Hong Kong, Shatin, N.T., Hong Kong.
Poon
Y T
YT
Wong
T M
TM
Mock
J W
JW
Ng
T B
TB
Wong
R N
RN
Yao
Q Z
QZ
Yeung
H W
HW
D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000187:drug effects; D011817:Rabbits; D012269:Ribosomal Proteins; D054788:Ribosome Inactivating Proteins; D012270:Ribosomes / Q000187:drug effects; D012639:Seeds / Q000737:chemistry; D017421:Sequence Analysis; D017385:Sequence Homology
D010940:Plant Proteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D008345:Manganese; C039582:MMC protein, Momordica charantia; D006026:Glycoside Hydrolases; D054788:Ribosome Inactivating Proteins