澱粉磷解酶與D-酵素的交互作用與在植物代謝的可能角色

Abstract

澱粉磷解酶 (L-SP) 與D-enzyme都是參與澱粉代謝的重要酵素，一般認為L-SP與澱粉合成較為相關 (Tickle et al., 2009)，且L-SP經磷酸化後會改變與澱粉合成相關酵素SBEI與SBEIIb的結合能力，進而影響澱粉的生合成 (Tetlow et al., 2004b)；而推測D-enzyme主要是參與澱粉的降解過程 (Zeeman et al., 2010)。此外，研究發現大腸桿菌之D-enzyme (malQ) 與1,4-alpha-D-glucan phosphorylase (malP) 位於同一操作組上 (Goda et al., 1997)，說明了D-enzyme與SP可能同時有基因表現並共同作用。張世宗 (1999) 在純化蛋白質過程，發現L-SP高分子量活性色帶HX，後續林怡岑發現D-enzyme也有往高分子量位移的現象。另外，由免疫共沉澱結果推測L-SP與D-enzyme可能有交互作用的現象 (林怡岑, 尚未發表)。 本研究乃以L-SP與D-enzyme的交互作用為主軸，先以GST pull-down assay及Far Western確認L-SP與D-enzyme可互相結合，再利用native/SDS 2D-PAGE與膠體過濾法分析，發現HX之次單元體組成可能為四個L-SP及四個D-enzyme次單元體，整體分子量約686 kDa。此外，比較添加磷酸酶抑制劑或CIAP (calf intestine alkaline phosphatase) 對HX形成之影響，發現磷酸化修飾可促進L-SP與D-enzyme結合。酵素活性分析結果顯示，形成HX蛋白質複合體後，大幅提升了D-enzyme活性，且擴大可利用的受質範圍。推測HX作用模式可能先以D-enzyme與受質反應後，再將產物繼續給L-SP進行磷解反應。進一步在不同生長時期的甘藷塊根中，觀察到L-SP含量與甘藷塊根中澱粉的充實程度成正相關，顯示L-SP可能與澱粉合成途徑較為緊密相關；而D-enzyme則在甘藷塊根剛發芽時，其蛋白質的表現量與酵素活性都增加，顯示D-enzyme可能主要參與澱粉降解的途徑。有趣的是，HX則分別在甘藷塊根快速生長時期，與在甘藷塊根發芽後，都有較多的蛋白質量，推測在澱粉快速消長時，植物透過蛋白質後修飾或其他調控形成HX，協助回收可利用的葡聚醣，以利澱粉代謝的進行。

Starch phosphorylase (L-SP) and D-enzyme are two important enzymes in starch metabolism. Several groups showed that L-SP is involved in starch biosynthesis (Tickle et al., 2009). L-SP can be phosphorylated, and the phosphorylated L-SP may form protein complexes with SBEI and SBEIIb which then affect the biosynthesis of starch (Tetlow et al., 2004b). On the other hand, it is believed that D-enzyme is mainly participated in starch degradation (Zeeman et al., 2010). Besides, it was found that D-enzyme (malQ) and 1, 4-alpha-D-glucan phosphorylase (malP) are encoded by the genes of the same operon in E. coli (Goda et al., 1997), suggesting that D-enzyme and SP may work together. During the purification process of L-SP, Chang (1999) found a high-molecular weight band (HX) showing L-SP activity. Subsequently, Lin also found that D-enzyme showed an extra form of catalytic activity having high molecular weight during its purification. The interaction of D-enzyme and L-SP was confirmed by co-immunoprecipitation (Lin, unpublished observation). In this study, GST pull-down assay and Far Western were utilized to confirm the protein-protein interaction between L-SP and D-enzyme. It was found that HX may be composed of four L-SP and four D-enzyme subunits, and the molecular weight of HX was estimated as 686 kDa by gel filtration and native/SDS 2D-PAGE. In addition, we checked the effect of phosphorylation/dephosphorylation on the formation of HX, and found that post-translational phosphorylation may promote the interaction of L-SP and D-enzyme. Moreover, high molecular weight form (HX) of D-enzyme demonstrated an enhanced catalytic activity, and showed wider range for various substrates. Therefore, in the protein complex (HX), D-enzyme might play the role in producing the malto-oligosaccharides which were then acted by L-SP via phosphorolysis. Furthermore, the protein level of L-SP increased when sweet potato roots accumulating starch, but decreased when sweet potato roots sprouted. This result showed that L-SP might be closely related to starch biosynthesis. However, the enzyme activity and protein expression of D-enzyme increased when sweet potato roots was about to sprout, indicating that D-enzyme might be mainly involved in starch degradation. Interestingly, the protein level of HX increased at two physiological stages: firstly, when the starch synthesis was active; and secondly, after sweet potato roots were germinated. These results suggested that the formation of HX might go through post-translational modification, which may then facilitate the starch metabolism by recycling the available glucans.