Mungbean (Vigna radiate L.) Starch Branching Enzyme Activity-Related Proteins in SDS-PAGE Gel after Renaturation

Abstract

成長中綠豆的溶解性萃取物(soluble extract)及成熟乾豆所製備的生澱粉顆粒(raw starch granule)，以SDS-PAGE電泳分析蛋白質圖譜後，藉由溶液萃洗膠條方法配合放射性活性測試法，篩選出具有澱粉分支酵素(starchbranching enzyme;SBE)活性的蛋白質，也藉由原位萃洗膠片方法配合活性染色法，在膠片上顯影出與SBE酵素活性相關的蛋白質。兩種方式均將變性蛋白質的SDS除去後，回復蛋白質構形與活性，同時粗估候選酵素蛋白質分子大小。SDS-PAGE電泳後的膠片按照分子量範圍作區分進行活性篩選，發現綠豆B7溶解性萃取物分子量在45-59kDa的區分，D35與D7品種生澱粉上之GBPs(granule bound proteins)分布在55-99kDa的區分，含有萃洗出SBE之高比活性，分別為B7是控制組的53倍(26.6U/mg)，D35是控制組的206倍(103U/mg)，以及D7是控制組的106倍(53U/mg)，顯示具有SBE活性之蛋白質被高度純化並且存在這些區分中。當針對55-99kDa區分當中的85、64、61及58kDa蛋白質以同法作萃洗追蹤，發現64kDa蛋白質萃洗出的比活性最高為611U/mg。並且發現85kDa蛋白質對於D35品種綠豆，64kDa與58kDa蛋白質對於D7品種綠豆的SBE活性有顯著的貢獻。原位萃洗SDS-PAGE膠片繼而活性染色的結果，顯示被碘染出的其中兩個條紋位置剛好相對應於以上的85kDa與64kDa蛋白質，因此，這兩個蛋白質極可能是在澱粉顆粒上與SBE活性相關的的蛋白質。A soluble fraction prepared from premature mungbean and extracts of raw starch granules from mature dry beans were analyzed by SDS-PAGE electrophoresis for protein profiles. Gel proteins were extracted by a solvent method and then screened for starch branching enzyme (SBE) activities by radioactivity assay. Gels were also subjected to in situ washing treatment followed by activity staining to visualize possible SBE activity-related proteins. The two different approaches both successfully removed SDS from denatured proteins and reconstituted protein conformation as well as activity while estimating the molecular weights of the candidate enzymes at the same time. The 45-59 kDa range of the B7 soluble fraction and the 55-99kDa range of the GBPs of both D35 and D7 fractions had high specific activity (S.A.). The S.A. of the B7soluble fraction was 53 times higher (26.6 U/mg), the D35 was 206 times higher (103 U/mg) and D7 was106 times higher (53 U/mg) than the control sample. The results indicate that renatured gel proteins regain their SBE activities and are highly purified following the solvent method. By tracing the SBE activities of the 85, 64, 61,and 58 kDa proteins within the active 55-99 kDa range, we found that the 64 kDa protein exhibited the highest S.A. (611 U/mg). Besides, the 85 kDa proteins of D35, and the 64 kDa and 58 kDa of D7, significantly contribute to SBE activity. In situ washing treatment followed by iodine activity staining showed two protein bands corresponding to the 85 and 64 kDa proteins, indicating that these two proteins are possible SBE activity-related proteins on mung bean raw starch granules.