| Summary: | 碩士 === 國立臺灣大學 === 植物學研究所 === 89 === SPLTI (sweet potato leaf trypsin inhibitor) was drought-induced in sweet potato leaf. The previous study of SPLTI found that it belongs to Potato I family. It is different since the reactive sites of SPLTI might be -R-E- which is obtained by primary sequence alignment and three dimensional (3-D) structural modeling. The molecular weight of SPLTI was 14kDa when analysis by SDS-PAGE. It is twice of the molecular weight of 7kDa deduced from amino acid sequence. No apparent disulfide bond was found through molecular modeling analysis. The objectives of the thesis were (1) to demonstrate the relationship of TI (trypsin inhibitor) activity and the reactive site, -R-E-; (2) to study whether the inter-molecular disulfide bonds contribute to dimerization of SPLTI. When using site-directed mutagenesis as the tool, it is proved that R46M and E47K mutated protein lost the TI activity, while C42S did not. The protomer of SPLTI can be separated when treated with -mercaptoethanol and dithiothreitol, respectively. The contribution of disulfide bond to dimerization of SPLTI was demonstrated by C42S and C59S mutated protein as well. The SPLTI and R46M/E47D double mutated protein did not show inhibitory activity to chymotrypsin and subtilisin. The equilibrium dissociation constant (Ki) of SPLTI to trypsin was 0.81(+/-)0.24 nM, C42S mutated protein to trypsin was 1.73(+/-)0.26 nM, and C59S mutated protein to trypsin was 1.64(+/-)0.24 nM. The importance of dimerization and the specific inhibitory activity of SPLTI were discussed.
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