利用化學聯結法探討水稻懸浮培養細胞中與蔗糖合成酶具交互作用之蛋白質

Abstract

蔗糖合成酶 (sucrose synthase, SuS) 在植物體中催化蔗糖和 UDP 形成果糖和 UDP-glucose 之可逆反應，此酵素對於在多醣類合成時的基質供應，以及在植物發育上都扮演重要角色。本論文利用化學聯結劑disuccinimidyl tartarate (DST)，探討水稻懸浮培養細胞 (Oryza sativa L. cv. Tainung 67) 中與蔗糖合成酶 (rice sucrose synthase, RSuS) 具交互作用之蛋白質。以 anti-RSuS 抗體進行西方點墨法的結果顯示，有數個與 RSuS 具聯結之蛋白質複合體存在，進一步以膠體過濾層析純化，並以 anti-RSuS 抗體進行免疫共沉澱後，所得之蛋白質進行 MALDI-TOF-MS 分析。在 in vitro 化學聯結實驗中觀察到的一些蛋白質複合體，在 in vivo 化學聯結實驗中存在類似結果，顯示在 in vivo 狀態下，RSuS 與其它蛋白質具交互作用。

Sucrose synthase (SuS) catalyzes the reversible interconversion of sucrose and UDP into fructose and UDP-glucose in plants. The enzyme plays an important role in providing substrates for polysaccharide synthesis and is crucial for plant development. To investigate whether SuS interact with other proteins, the chemical cross-linker, disuccinimidyl tartarate (DST), was used to covalently cross-link the interacted proteins in rice suspension-cultured cells of rice (Oryza sativa L. cv. Tainung 67). Western analysis with anti-RSuS antibody showed the presence of several cross-linked protein complexes containing RSuS. The RSuS-containing protein complexes were partially purified by gel filtration chromatography and immunoprecipitated with anti-RSuS antibody, and then subjected to MALDI-TOF-MS analysis. The in vivo cross-linking experiment revealed that some RSuS-containing different protein complexes observed in the in vitro cross-linking experiment were also present in the in vivo cross-linked proteins of rice suspension-cultured cells, suggesting that the interactions between RSuS and other proteins occur in vivo.