Partially purified mungbean ( Vigna radiata var. KPS1) soluble fractions with starch branching enzyme (BE) activity detected by radioactive method were subjected to Native-PAGE and in situ activity staining for enzyme analysis. When native gel was incubated with G-1-P and sodium citrate (pH 7.0) to react at 30 ？HJ for 2 hrs followed by iodine staining, it visualized a blue-purple protein population with slow mobility. SDS-PAGE and silver staining showed that it was consisted of 105, 62 and 55 kDa proteins. The polyclonal antibody against the 62 kDa protein was able to immuno-neutralize 97% SBE activity and cross-react majorly with 62, 55 and 31 kDa proteins of mungbean crude extract by Western blotting. The polyclonal antibody against the 55 kDa protein also cross-reacted with 62, 55 and 31 kDa proteins. Tryptic peptide mapping and database searching for the 105 kDa protein demonstrated it to be low-affinity starch phosphorylase (L-SP) due to its high internal sequence homology with L-SP of sweet potato, corn, rice and potato. It suggested that mungbean L-SP is associated with 62 kDa and 55 kDa protein at native state. When native gel was incubated in the presence of phosphorylase a and AMP in addition to G-1-P and sodium citrate (pH 7.0) to react at 30 ？HJ for 21 hrs, two red-purple protein populations with high mobility were visualized by iodine staining, which was similar to the finding of rice BEIIa and BEIIb. Developing mungbeans of different seed size were analyzed by the phosphorylase-stimulated activity staining method and found that the density of SP-containing slow mobile population decreased and the high mobile population increased as the seed size increase. These results demonstrated that various mungbean BE isoforms may play important roles in starch accumulation during seed development.
