Identification of starch branching enzyme in mungbean

Autor: Jin-Yi Chang, 張敬宜
Rok vydání: 2002
Druh dokumentu: 學位論文 ; thesis
Popis: 90
Mungbean (Vigna radiata, cv. KPS1) soluble extract (B7 crude) was subjected to Native-PAGE followed by an in situ activity staining of phosphorylase-stimulated starch branching enzyme (SBE) that three SBE populations were visualized. One blue-purple protein population with low mobility was denominated as SBEI, and two other red-purple protein populations with high mobility were denominated as SBEIIa and IIb. SBEI band appeared on gel slab after reacting for 2 hrs, whereas SBEIIa band emerged after 16 hrs. SDS-PAGE and silver staining showed that SBEI was consisted of 105, 62 and 55 kDa proteins and SBEIIa was consisted of 96, 64 and 55 kDa proteins. B7 crude was partially purified for SBE activity by a series of liquid chromatography steps. It was apparently resolved into peak PI and PII fractions by Sephacryl S-200 column chromatography. Activity staining showed that PI fractions belong to blue-purple SBEI population, and PII fractions belong to red-purple SBEIIs population. Each of the pooled PI and PII fractions were separated by Q2 column chromatography and purified into peak PIII with 30.4 purification fold and peak PIV with 101.9 purification fold respectively. SDS-PAGE analysis of peak PI and PIII indicated that the intensity of 105, 62 and 55 kDa proteins were correlated with SBEI activity and that of 96, 64 and 55 kDa proteins were correlated with SBEII activity. Trypsin digestion and MALDI-TOF mass analysis for the 105 kDa protein demonstrated it to be starch phosphorylase(SP) due to its high internal sequence homology with sweet potato, maize and rice. Polyclonal antibodies against the 62 kDa protein were able to immuno-neutralize 97% SBE activity and cross-react mainly with 62, 55 and 31 kDa proteins of B7 crude by Western blotting. Polyclonal antibodies against the 55 kDa protein also immuno-neutralized SBE activity but cross-reacted with 62, 55 and 31 kDa proteins. The overall above evidence suggested that 62 and 55 kDa proteins belong to the SBEI population, and they are associated with 105 kDa protein at native state. Developing mungbean samples of three different seed size were analyzed by the phosphorylase-stimulated SBE activity staining assay and found that as the seed size increase, the SBE specific activity and the density of SBEI population decreased, but the density of SBEIIa population increased. SDS-PAGE and silver staining showed that the B7 crude fractions of these three samples have comparable major protein profiles and their 62 and 55 kDa protein contents were similar. Besides, SBE activity was measured by a colorimetric method based on analysis of the reaction products demonstrated that PI fractions showed both SP and SBE activity, but PII fractions revealed majorly SBE activity. This fact was again proven by an amylose-branching assay of the PI and PII fractions that the maximum absorbance of amylose(blue-blank) at 600-650 nm decreased that comes along with the absorption spectrum profile of reaction product shifted to amylopectin (red-purple) at 550 nm. These results indeed demonstrated that substrate amylose was transformed into amylopectin product by SBEs in these active fractions. In conclusion, the above studies disclosed that there are at least three mungbean SBE isoform populations which play important roles in starch biosynthesis during seed development.
Databáze: Networked Digital Library of Theses & Dissertations
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