甘藷塊根澱粉磷解脢高溫下階段式降解之探討

The Studies of Proteolytic Degradation of L-form Starch Phosphorylase from Sweet Potato Roots

葉昭圻(Chou-Chi Yeh)

指導教授 ： 莊榮輝

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00744

摘要

植物的L型澱粉磷解脢 (L-form starch phosphorylase, L-SP) 在其分子中央，較動物的肝醣磷解脢多出一段由78個胺基酸組成的片段 (L78)。因為胺基酸序列及結構特殊的關係，L78極易斷裂，造成L-SP分子裂解成兩群分子量 (約50 kD) 相近的片段 (統稱F50s)。將甘藷塊根切片加熱至45℃ 以上會使L-SP之泳動率改變，產生規律之階段式降解；隨著加熱反應時間的增加，Anti-L78P抗體最早失去辨識L-SP的能力，因此推論降解過程乃先移除中央之L78；接著J3b無法辨識L-SP，因此降解由L78繼續朝N-端方向進行，而H7c結合的區域 (L-SP之C-端) 較少發生降解。在甘藷塊根加熱過程中，當溫度與濕度到達一定程度，甘藷塊根切片便產生糊化現象：由掃描式電顯得知，溫度處理造成澱粉粒破裂。此外，由切片上不同區域的原態電泳檢定，發現糊化與L-SP的降解有正相關性：且隨著甘藷切片糊化程度的不同，L-SP產生階段式降解。因此我們推測，甘藷切片在高溫處理下，內部的澱粉粒因糊化而破裂，當胞器破裂後，L-SP被其他酵素降解機會增加，因而產生所觀察到的階段式降解。

關鍵字

澱粉磷解脢

並列摘要

In the middle of plant L-form starch phosphorylase (L-SP), a 78-amino acid insertion (L78) is found when compared with its counterpart in animal cells, glycogen phosphorylase. L78 is susceptible to multiple proteolytic cuttings because of its unique amino acid sequence and molecular structure.It was found that L-SP is cleaved into two groups of fragments with the molecular mass around 50 kD (F50s). When the sweet potato roots were heated at 45℃, the mobility of L-SP was changed on native-PAGE gel electrophoresis. The immunostaining pattern using Anti-L78P as the probe revealed that heating induced the cleavage of L-SP molecules on the L78 initially. Part of the N-terminal half of L-SP was then cleaved, but the C-terminal part remained intact. Although L-SP was proteolytically modified , it still remained its native structure as well as the enzymatic activity. The heating process induced the gelatinization of the sweet potato root slice when the temperature and humidity achieved the optimal condition. The gelatinization of sweet potato roots that caused the collapse of starch granules was confirmed by SEM. In addition, it was found that the sequential degradation of L-SP was correlated with the gelatinization by comparing the L-SP from gelatinized and un-gelatinized tissues. Therefore, we conclude that heat treatment induce the gelatinization inside sweet potato roots. This process accelerates the breaking down of the amyloplast, which might correlates with the sequential degradation of the plastidal enzyme, L-SP.

並列關鍵字

Starch Phosphorylase

參考文獻

張世宗 (1999) 甘藷塊根Chaperonin及Proteasome之分離與性質研究　博士論文 國立台灣大學 台北

曾光靖 (2005) 磷酸化修飾對甘藷塊根L型澱粉磷解脢之影響 碩士論文 國立台灣大學 台北

楊光華 (2005) 甘藷塊根澱粉磷解脢激脢之純化與性質分析 博士論文 國立台灣大學 台北

Alfonso M, Yruela I, Almarcegui S, Torrado E, Perez MA, Picorel R (2001) Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr. Cell cultures deficient in fatty acid desaturation. Planta 212: 573–582

Alison M. Smith, Samuel C. Zeeman, David Thorneycroft and Steven M. Smith (2003) Starch mobilization in leaves. Journal of Experimental Botany 54: 382.