綠豆質子泵焦磷酸水解酶(VrH+-PPase)屬於膜鑲嵌焦磷酸水解酶(M-PPase)(EC 3.6.1.1)，離子泵焦磷酸水解酶存在於大部分植物中，也存在於一些藻類、原生動物、細菌、古細菌，但酵母菌與動物則不具有此類蛋白。VrH+-PPase主要表現於綠豆增生組織的酵素，在調節細胞中焦磷酸濃度扮演很重要的角色，焦磷酸是合成巨分子(DNA、RNA、protein、aminoacyl-tRNA、cellulose)、糖質新生與蔗糖合成的副產物，焦磷酸會調控這些反應，是植物成熟的一關鍵蛋白。而質子泵焦磷酸水解酶(H+-PPase)分為鉀離子依賴性與獨立性兩種次家族，而轉運的陽離子也分為質子或鈉離子以及兩種兼具的次家族。VrH+-PPase是屬於鉀離子依賴性，轉運的離子為質子。在先前VrH+-PPase的分子結構中，確定了具16個穿膜螺旋結構(TMH)，其中TMH 5/6/11/12/15/16形成的質子轉運通道的核心。並指出R242/D294/K742/E301為質子轉運途徑的關鍵胺基酸，在通道中的一疏水性閘門L232/A305/L555/V746，以及通道出口E225/T228/I312/R562/P753均扮演重要的角色。此研究中進行點突變E301Q、L555M/K、E225H/S/A去分析質子轉運能力。結果顯示E301Q和L555K突變蛋白完全失去質子轉運能力，推測因E301Q失去了帶電性而失去質子轉運能力，而在先前研究L555K的結構中發現鎖住的水分子推測因此失去了活性。L555M轉運能力約剩20%，推測是疏水性閘門縮小所導致質子轉運能力下降。E225H和E225S約剩80%，E225A約剩50%質子轉運能力，推測是影響了重要E225-R562之間的鹽橋所造成的質子轉運能力下降。

Vigna radiata H+-translocating pyrophosphatase (VrH+-PPase) belongs to the class of membrane-embedded pyrophosphate hydrolase (M-PPase) (EC 3.6.1.1). The H+-translocating inorganic pyrophosphatase distributed among most plants, but only some alga, protozoa, bacteria, and archaebacteria. The VrH+-PPase major express in mung bean germination and it is a potassium-dependent proton transporter. The three-dimensional structure of VrH+-PPase has been determined in our laboratory. consisting of 16 transmembrane helices (TMHs) and TM5/6/11/12/15/16 formed the core which are in charge of the pyrophosphatase hydrolysis and proton pumping. Previous studies indicated that R242/D294/K742/E301 are the key residues in the proton transport pathway. Along the ion transport pathway, a hydrophobic gate, L232/A305/L555/V746 and a channel exit, E225/T228/I312/R562/P753 also play essential roles in VrH+-PPase. In this study, we studied mutants of E301Q, L555M/K at the hydrophobic gate and E225H/S/A at the exit channel. E301Q and L555K mutants completely lost the proton pumping ability and it suggest that E301Q lost its negative charge and lost its ability to transport proton. The order waters were found at the hydrophobic gate of L555K that it may abolish its proton pumping. L555M mutant preserved 20% proton pumping because methionine narrow down the space of the hydrophobic gate. E225H and E225S as well as, E225A keep about 80% and 50% proton pumping ability, respectively and it presumably because of the breaking the salt bridge E225-R562.

目錄
中文摘要 I
Abstract II
誌謝 III
目錄 IV
第一章簡介 1
1.1焦磷酸水解酶分型 1
1-2膜鑲嵌質子泵焦磷酸水解酶在植物生理之角色 2
1-3綠豆質子泵焦磷酸水解酶結構 3
第二章實驗材料與方法 5
2.1酵母菌轉殖作用(transformation) 5
2-2放大與誘導酵母菌表現膜蛋白VrH+-PPase 5
2-3溶解酵母菌的細胞壁 6
2-4製備微粒體(純化) 6
2-5純化膜蛋白VrH+-PPase 7
2-6晶體設置 8
2-7製備含有VrH+-PPase的液泡膜 8
2-8水解活性分析 10
2-9質子轉運活性分析 10
2-10 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳與西方墨點法 11
2-11布拉德福蛋白質定量法(Bradford protein assay) 12
第三章結果 14
3-1製備VrH+-PPase的液泡膜條件 14
3-2 pH值對於VrH+-PPase質子轉運能力的影響 15
3-3第555號胺基酸(亮胺酸)單點突變之質子轉運能力與水解活性分析 15
3-4第301號胺基酸(麩胺酸)單點突變之質子轉運能力與水解活性分析 16
3-5第225號胺基酸(麩胺酸)單點突變之質子轉運能力與水解活性分析 17
3-6 E301Q-PPi晶體複合體 17
3-7 E225A-PPi晶體複合體 18
第四章討論 19
4-1疏水性閘門L555之角色 19
4-2質子轉運區E301之角色 20
4-3通道出口E225之角色 20
4-4晶體培養情況之探討 21
圖表 22
參考文獻 38

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