The identification of starch phosphorylase in the developing mungbean (Vigna radiata L.).
Starch phosphorylase (SP) in immature mungbean (Vigna radiata L. cv KPS1) seed soluble extract was detected by in situ activity staining and identified by MALDI-TOF mass analysis. After in situ SP assay on native-PAGE, a major starch-enzyme complex was located on the gel zymogram in a dose-dependent manner. This complex depicted two major SP-activity related proteins, 105 kDa and 55 kDa, by SDS-PAGE. The mass and predicted sequence of the tryptic fragments of the isolated 105 kDa protein, analyzed by MALDI-TOF spectroscopy and bioinformatic analysis, confirmed it to be mungbean SP as a result of high similarity to the L-SP of known plant. Polyclonal antibodies raised from the 55 kDa recognized both the 105 kDa and the 55 kDa proteins on the Western blot and neutralized partial SP activity, indicating that the two proteins were immunologically related. The 55 kDa protein possess high similarity to the N-terminal half of the 105 kDa SP was further confirmed. The SP activity and the activity stained protein density in mungbean soluble extract decreased as the seed size increased during early seed growth. These data indicate that mungbean 105 kDa SP and SP activity-related 55 kDa were identified in the developing mungbean.
Ko
Yuan-Tih
YT
Department of Food Science, Biotechnology Division, College of Life and Resource Sciences, National Taiwan Ocean University, 2 Pei-Ning Rd., Keelung 20224, Taiwan.
Chang
Jin-Yi
JY
Lee
Ya-Ting
YT
Wu
Yi-Hui
YH
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology* / Q000254:growth & development*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012639:Seeds / Q000201:enzymology* / Q000254:growth & development*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D024941:Starch Phosphorylase / Q000032:analysis* / Q000737:chemistry
D010446:Peptide Fragments; D024941:Starch Phosphorylase