Proteasome是真核細胞中重要的蛋白質水解系統,它可以降解被ubiquitin標定的蛋白質,藉以維持細胞的正常功能。澱粉磷解脢 (L-SP) 在其胺基酸序列上,具有PEST region及destruction box等訊號序列,顯示容易遭受降解;而研究也發現L-SP的確容易降解,並可能與其活性調節有關 (Chen et al., 2002)。張世宗 (1999) 在純化L-SP的過程中,發現一高分子量L-SP活性色帶HX,並推測HX可能是L-SP和proteasome的結合體。陳安娜 (2001) 續以親和層析管柱的專一性吸附能力,證明L-SP與proteasome可能相互結合。林怡岑 (2003) 以雙向免疫擴散,證實HX確是L-SP與proteasome的結合體。本論文接續上述研究,在HX的純化樣本中發現L-SP與proteasome的活性區重疊;HX以原態膠體電泳之免疫呈色結果發現,J3b抗體無法與L-SP結合,此種遮蔽現象卻可在SDS-PAGE之免疫呈色中解除,顯示proteasome可能結合在L-SP的N-端。利用Blue native PAGE加上LC-MS/MS的身分鑑定,確定HX為L-SP與proteasome的蛋白質複合體。已知目標蛋白質的降解,可透過磷酸化修飾與proteasome結合,但實驗顯示成熟甘藷塊根中,所含磷酸化L-SP極少,其詳細機制有待進一步探討。
Proteasome is a controlled proteolytic system in eukaryotic cell, which degrades proteins recognizied and modified by ubiquitins. This ubiquitin-proteasome system plays important physiological roles, and might regulate the activity of proteins or enzymes in the cell. On the molecule of L-form starch phosphorylase (L-SP), several PEST regions and destruction boxes were found, indicating that it was subjected to degradation. Chen et al. (2002) showed that this proteolytic modification might play a regulatory role in controlling the direction of the L-SP catalysis. On the native electrophoresis gel for L-SP activity during the purification of L-SP, Chang et al. (1999) have found a high-molecular-weight band (HX) showing L-SP activity, which contains both proteasome and L-SP. Chen (2001) further analyzed the composition of HX by the affinity absorbent using specific antibodies against proteasome or L-SP, which was also confirmed by Lin (2003) using double diffusion. This study further improved the purification procedure for HX by tracing the overlaping activity fractions for L-SP and proteasome. The binding of J3b antibody to its epitope on L-SP was blocked by proteasome, as observed by native-PAGE immunoblotting. However, this blocking of antibody binding was relieved on SDS-PAGE immunoblot indicating that L-SP might be bound to proteasome in its N-terminal half. The HX protein was further analyzed by Blue native-PAGE and LC/MS/MS to reveal its components as L-SP and proteasome. The ubiquitin- and proteasome-dependent proteolytic pathway might take phosphorylated protein as one of its targets. However, we have only detected very small amount of phosphorylated L-SP in sweet potato roots. Further study is needed to elucidate the physiological function of the phsophorylation of L-SP and its association with the ubiquitin-proteasome system.
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