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研究生:石立杰
研究生(外文):Lie-chieh Shih
論文名稱:轉移核醣核酸鳥糞嘌呤鹽基-Q鹽基轉醣酵素之研究
論文名稱(外文):Study on tRNA-guanine transglycosylase
指導教授:吳榮燦
指導教授(外文):Rong-Tsun Wu, Ph. D.
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:70
中文關鍵詞:轉移核醣核酸鳥糞嘌呤鹽基Q鹽基轉醣酵素蛋白質激 CK562 細胞株
外文關鍵詞:tRNARNA transferguanineGqueuinetransglycosylasePKCprotein kinase Ccalcium depandent protein kinaseK562 cell line
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真核生物的轉移核醣核酸鳥糞嘌呤鹽基-Q鹽基轉醣酵素( tRNA-guanine transglycosylase,簡稱 TGT )(EC 2.4.2.29)為在細胞中負責催化轉移核醣核酸於轉錄後在反密碼(anticodon)上進行Q鹽基超修飾之酵素。此超修飾現象發生於天門冬酸、醯胺天門東酸、酥胺酸及組織胺酸等四組轉移核醣核酸上;其作用為將鳥糞嘌呤鹽基置換為Q鹽基。此種Q鹽基超修飾現象的生理意義至今仍不甚明瞭,但已知與細胞分化及腫瘤的惡性程度有密切關連:在分化程度低的細胞及腫瘤惡性程度越高的細胞中,轉移核醣核酸反密碼上之Q鹽基超修飾現象的程度越低。反之,在正常已分化的細胞中,則普遍出現轉移核醣核酸反密碼上有Q鹽基超修飾現象。
本實驗室已建立一純化此酵素的步驟,並以此步驟為基礎改良純化此酵素的方法;利用超高速離心、硫酸銨沈澱、分子篩凝膠管柱(TSK HW 55F)、等電點電泳聚焦(Rotofor IEF)、及Heparin親和性管柱來做初步分離,最後以聚丙烯胺凝膠電泳(Prep Cell)將酵素之次單位依分子量大小沖提出,而得到了高純度的TGT 60kDa次單位。並將純化所得之TGT 60kDa次單位混合Freund's佐劑注射於兔子皮下以取得免疫抗體。
利用所得能辨識酵素60kDa次單位之專一性多株抗體製作能吸附TGT酵素之親和性凝膠。將初步純化之去免疫球蛋白污染的TGT檢體與親和性凝膠作用,在未吸附之引流液中檢測酵素活性為1.9 pmole/h 100ml,在酸性沖提液所沖提出之檢體在平衡酸鹼值及透析後則可檢測出5.8 pmole/h 100ml之活性;而此檢體在經復活反應10天後其活性可達11 pmole/h 100ml。以電泳分析此檢體成分與TGT酵素之次單位組成相同。
以此專一性抗體進行免疫細胞染色偵測TGT酵素在Ara-C誘導分化之K562細胞中的表現;發現細胞核螢光亮度增加,螢光亦出現於細胞質中。此結果說明此酵素在K562細胞進行Ara-C誘導分化時有表現。另外本研究也將TGT酵素次單體,經胰蛋白脢(trypsin)作用成小片段之peptide,並將分離純化之peptide進行胺基酸定序。

The eukaryotic tRNA guanine transglycosylase (TGT, E.C.2.4.2.29) catalyzes the post-transcriptional modification, queuine modification, on the anticodon of the tRNAs in the cell. The replacement of guanine into queuine is specific for 4 species of tRNAs: Aspartyl-, Asparaginyl-, histidinyl-, and tyrosinyl-tRNAs. The physiological significance for the queuine modification remains unknown, however, the amount of the resulting queuosine nucleotide (Q) is dependent on the stage of differentiation and the malignance grade of tumor. Neoplastically transformed and hyper-proliferative cells usually have queuine hypomodification. Whereas in normal cells have queuine modification.
In this study, the TGT enzyme was purified by a modified procedure based on previous established procedure in our laboratory. By ultracentrifugation, ammonium sulfate precipitation, TSK HW 55F gel filtration chromatography, Rotofor isoelectric focusing, Heparin affinity chromatography, and Prep Cell SDS polyacryamide gel electrophoresis, the TGT 60kDa subunit is purified to near homogenity. The purified TGT 60kDa subunit were mixed with Freund's adjuvant and immunized to rabbit for production of polyclonal antibody.
The TGT enzyme could be adsorbed by this polyclonal antibody conjugated with CNBr activated sepharose gel. The sample eluted by the acidic elute buffer could retain TGT activity within 5.8 pmole/h 100ml, compared with sample of 1.9 pmole/h 100ml that was not adsorbed by the gel. After renaturation, the eluate had a higher enzyme activity of 11 pmole/h 100ml. Electrophoresis analysis showed the component of the eluted sample is the same as TGT subunit.
Immunocytochemistry study on the expression of TGT enzyme during Ara-C triggered K562 cells differentiation. The result shows the increase of fluorescence in the cell nuclear in the differentiated cells and the fluorescence also presents in the cytoplasm. The result indicates the expression of TGT is induced during K562 cell differentiation. In another study, TGT 60kDa subunit was treated with trypsin, and the resulting peptides were purified for subsequent N-terminal amino acid sequencing.

中文摘要 ………………………………………………………1
Abstract . ……………………………………………………...3
緒論 …………………………………………………………….5
試藥與器材 …………………………………………………..10
實驗方法 ……………………………………………………..17
實驗結果 ……………………………………………………..25
討論 …………………………………………………………...29
圖表 …………………………………………………………...35
References …………………………………………………...62

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