臺灣博碩士論文加值系統

低澱粉親和型之澱粉磷解脢 (L-SP) 廣泛存在於植物澱粉貯藏組織中，在in vitro情況下，發現有三種不同催化方式︰(1) 需醣引子的直鏈醣合成反應、(2) 不需醣引子之合成直鏈醣 (primer-independent activity, PI activity)、(3) 磷解澱粉反應。L-SP也有幾種不同的降解型式︰(1) 完整的L-SP含有特殊的L78片段、(2) L78部份斷裂的L-SP (L-SP’)、(3) L78完全斷裂的L-SP (L-SP*)。本論文釐清各種修飾形式的L-SP各自扮演何種功能︰當L-SP為完整的110 kDa時，不需醣引子直接以Glc-1-P合成直鏈醣，而SP* 就失去此種PI活性。另外，完整的L-SP及L78斷裂不完全的L-SP’，都可使用直鏈寡葡萄醣 (G2, G3, G5, G7, 數字代表葡萄糖單位) 進行直鏈醣合成，但L78若完全去除成為SP*，就無法使用較短的寡醣 (G2, G3)；而其中L-SP利用短鏈寡醣 (G2, G3) 與長鏈寡醣 (G5, G7) 合成直鏈醣的機制也不相同。在L-SP合成直鏈醣的反應中，速率決定步驟為從單醣Glc-1-P合成雙醣 (G2)。另一方面，L-SP藉由LSK磷酸化，加速L78的斷裂，可對上述L-SP三種反應巧妙調節；造成此斷裂的最後因子為蛋白脢，但此種降解模式為可調節性，並且不影響L-SP的構型，仍維持著完整活性。另外，也利用native-PAGE/SDS-PAGE及LC-MS/MS分析LSK，進行LSK peptide合成以製備LSK的單株抗體，將可進一步了解L-SP的催化機制及澱粉代謝上所扮演的生理角色。

The low-affinity type of starch phosphorylase (L-SP) is widely found in the starch-accumulating tissues of plant. In the test tube, it shows three types of catalysis: (1) the biosynthesis of oligo-glucan in the presence of a primer; (2) the same biosynthetic reaction in the absence of a primer (primer-independent activity, PI activity); and (3) the degradation of starch by phosphorolysis. The purified L-SP might have three modified forms: (1) the intact 110 kDa molecule containing the L78 insertion in the middle of L-SP; (2) the modified L-SP in which the L78 is nicked by proteolysis (L-SP’); and (3) the L78 on L-SP is essentially removed (L-SP*). Our study found that the modification on the L78 might cause the change of the catalytic behavior of L-SP. The intact L-SP can synthesize amylose directly from Glc-1-P in the absence of a prime. However, the SP* lost this PI activity completely. For amylose synthesis, the intact L-SP and SP’ can utilize various oligo-glucan (G2, G3, G5 and G7, numbers indicate units of glucose) as the primer; nevertheless, the SP* can not take G2 and G3 as the substrates. Furthermore, the catalytic mechanism of L-SP toward the shorter glucan (G2 and G3) was different from the longer glucan, G5 and G7. The rate-limiting step in the primer-independent reaction of L-SP was the formation of one disaccharide from two molecules of Glc-1-P. On the other hand, the L-SP was reported to be phosphorylated at a Ser residue on the L78 (Young et al, 2006). The phosphorylation of L-SP then enhanced the proteolytic modification of L78, which might accordingly regulate the enzyme behavior of L-SP between the three types of catalysis. The proteolysis of the phosphorylated L-SP might be regulated by an unknown mechanism. The specific kinase for the phosphorylation of L-SP was isolated by native/PAGE and SDS/PAGE, and its partial amino acid sequence was determined by LC/MS/MS. The antibody against a peptide from this kinase sequence was prepared by hybridoma technique, which might be useful for the exploration of the role of L-SP in starch biosynthesis.

目錄 I
中文摘要 IV
Abstract V
縮寫表 VI
第一章 緒論 1
1.1 澱粉磷解脢 1
1.1.1 澱粉磷解脢分類 1
1.1.2 植物澱粉磷解脢與動物肝醣磷解脢的比較 2
1.2 澱粉磷解脢的生理角色 2
1.2.1 磷解澱粉 3
1.2.2 合成澱粉 3
1.2.3 不需醣引子合成直鏈醣之活性 4
1.2.4 其他活性 4
1.3 澱粉磷解脢的調控機制 4
1.3.1 植物L型澱粉磷解脢與動物肝醣磷解脢的比較 4
1.3.2 L78對L型澱粉磷解脢的影響 5
1.4 澱粉磷解脢激脢 9
1.4.1 磷酸化修飾可調控蛋白質之生化性質 9
1.4.2 訊息傳導 9
1.5 研究動機 10
第二章 材料與方法 12
2.1 一般分析法 12
2.1.1 蛋白質定量法 12
2.1.2 甘藷塊根澱粉磷解脢L-SP合成澱粉活性分析法 (添加醣引子) 13
2.1.3 甘藷塊根澱粉磷解脢L-SP合成澱粉活性分析法 (不添加醣引子) 14
2.1.4 甘藷塊根澱粉磷解脢L-SP合成澱粉活性分析法 (添加直鏈短醣) 15
2.1.5 甘藷澱粉磷解脢激脢活性分析法 16
2.2 管柱色層分析法 17
2.2.1 管柱色層層析法之基本操作 17
2.2.2 膠體前處理與保存 19
2.2.3 膠體過濾法 19
2.2.4 離子交換法 20
2.2.5 疏水性層析法 22
2.2.6 Ni-NTA-agarose親和性層析法 22
2.3 電泳檢定法 23
2.3.1 原態膠體電泳 23
2.3.2 SDS膠體電泳 26
2.3.3 native-PAGE/SDS-PAGE膠體電泳 28
2.3.4 製備式電泳與電泳溶離 28
2.3.5 膠體染色法 30
2.3.6 膠片乾燥法及護貝 33
2.3.7 蛋白質電泳轉印法 34
2.4 免疫學方法 35
2.4.1 酵素免疫染色法 35
2.4.2 酵素免疫分析法 37
2.5 甘藷塊根L型澱粉磷解脢製備法 38
2.5.1 酵素粗抽取及硫酸銨分劃 38
2.5.2 甘藷塊根L型澱粉磷解脢純化法 40
2.5.3 甘藷塊根L型澱粉磷解脢激脢純化法 41
2.5.4 磷酸化修飾之澱粉磷解脢製備法 42
2.5.5 大腸桿菌表現蛋白質L78之誘導與純化 43
2.6 膠體內蛋白脢水解 45
2.7 單株抗體之製備 46
2.7.1 小白鼠免疫 46
2.7.2 細胞融合 47
2.7.3 細胞保存法 51
2.7.4 單株抗體的生產 52
2.7.5 免疫球蛋白之純化 52
第三章 結果與討論 54
3.1 各種酵素材料之製備 54
3.1.1 植物L型澱粉磷解脢製備法 54
3.1.2 製備不同L78降解程度之L型澱粉磷解脢 (L-SP*, L-SP’) 54
3.1.3 L78序列之表現及純化 54
3.1.4 甘藷塊根磷解脢激脢LSK之純化 55
3.2 L-SP合成直鏈醣之模式 62
3.2.1 L-SP不需外加醣引子仍可合成直鏈醣 62
3.2.2 L78序列參與不需醣引子合成直鏈醣之催化活性 62
3.2.3 證明L-SP合成直鏈醣確實不需醣引子存在 65
3.2.4 降解的澱粉磷解脢 (L-SP*, L-SP’) 所需最短醣引子長度 67
3.2.5 L-SP合成直鏈醣之機制 72
3.3 L-SP調控機制 87
3.3.1 L-SP的專一性磷酸化激脢LSK 87
3.3.2 LSK的生化性質 87
3.3.3 以LC-MS/MS鑑定LSK身分 87
3.3.4 LSK對L-SP磷酸化修飾的意義 92
3.4 LSK單株抗體之製備 97
3.4.1 抗原peptide之選擇及抗原製備 97
3.4.2 抗體之製備 97
3.4.3 抗體之篩選及專一性鑑定 97
第四章 結論 100
4.1 L78的斷裂有無影響L-SP活性的表現 100
4.2 L-SP的調控機制 100
4.3 L-SP的代謝機制 101
4.4 總結 101
4.5 未來展望 102
參考文獻 104

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