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研究生:蔡百騏
研究生(外文):Pai-Chi Tsai
論文名稱:利用Biacore系統探討一型蛋白磷酸水解酶和其抑制蛋白間的交互作用力
論文名稱(外文):Characterization of the interaction between PP1 and its inhibitory protein by surface plasmon resonance biosensors
指導教授:黃憲斌
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:一型磷酸水解酶乙型抑制蛋白
相關次數:
  • 被引用被引用:1
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乙型抑制蛋白( Inhibitor-2;I2 )藉由其多重結合位置來達到抑制第一型蛋白質磷酸水解酶 ( PP1 )的目的。在實驗室之前的研究指出,I2的胺基酸序列第九到十三個殘基 (PIKGI)對於抑制PP1活性上扮演重要的角色;相對於和大多數PP1的調控蛋白中所具有的BB(V/I)XF序列有著高度相似性的第一百四十四到一百四十七個殘基(KLHY),則缺乏明顯的證據指出此序列對於抑制PP1上的重要性。因此,在這裡我們針對於此序列 144KLHY147是否真正具有抑制PP1的功能,作一個深入的研究;在Biacore分析系統的研究之下,我們發現I2的突變蛋白I2-(1-120)、I2-(1-140)、I2-(1-160)、I2-(1-180)和PP1的結合能力平衡常數值隨著刪除C端胺基酸的數目增加而有稍微下降的趨勢,或許可以解釋其中的胺基酸序列144KLHY147有參與部分的結合;但是在I2點突變蛋白 (Y147A)的結合能力測試中,其平衡常數值和I2-(WT)幾乎相同,可提供直接的證據來的說明此序列144KLHY147並不參與抑制PP1的作用。
Inhobitor-2 (I2) is the regulatory subunit of the catalytic subunit of protein phosphatase-1 (PP1) and inhibits the enzyme activity via multiple sites. Our previous studies have demonstrated that the region encompassing residues 9-13 (PIKGI) plays critical role in inhibition of PP1. However, no obvious evidence indicated the sequence Lys144-Leu-His-Tyr147 corresponding to the BB(V/I)XF PP1-binding motif, involved in this action. In the presentation, we have characterized in detail the function of this sequence to test whether it has such activity. Biacore analysis demonstrated that deletion of C-terminal sequence after the residue 120 of I2 did not significantly affect the binding constant between PP1 and I2. In comparison with I2(WT), mutation of Tyr-147 【I2-(Y147A)】 also showed no significant difference on affinity with PP1. These findings suggested that this sequence Lys144-Leu-His-Tyr147 is probably not involved in the inhibition with PP1.
第一章 序論…………………………………………………………….. 1
1-1 背景介紹………………………………………………………….…. 1
1-2 實驗目的……………………………………………………….…...10
第二章 實驗材料與方法…………………………………………….… 17
2-1 實驗材料……………………………………………………….…...17
2-2 實驗儀器……………………………………………………….…...17
2-3 實驗方法……………………………………………………….…...20
2-3-1 緩衝溶液配製……………………………………………….…...20
2-3-2 重組型PP1的純化……...………………………………………..21
2-3-3 天然型PP1的純化…..……………………………………….…..22
2-3-4 利用生物素標定I2與I2突變蛋白…………………………….… 23
2-3-5 利用BIA系統研究分子間結合的動力學機制……………….....24
第三章 實驗結果………………………………………………...………27
3-1 BIA系統中解離溶液的選擇……….……………………………....27
3-2 BIA系統對於PP1與I2分子間結合的動力學機制…..…………… 27
第四章 討論……………………………………………………………...30
第五章 參考文獻………………………………………………………… 33
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