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研究生:沈樹林
論文名稱:利用Phenol誘導黃果蠅之GlutathioneStransferase的純化及特徵之研究
論文名稱(外文):Characterization and purification of glutathione s-transferases in drosophila melanogaster induced by phenol
指導教授:簡一治
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:科學教育研究所
學門:教育學門
學類:普通科目教育學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:33
中文關鍵詞:黃果蠅
外文關鍵詞:PhenolGlutathione Stransferase
相關次數:
  • 被引用被引用:0
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摘要(Abstract)
存於細胞質的glutathione S-transferase,可催化glutathione和各式不同外來化學物質(xenobiotics)所形成的連結物(conjugations),包括殺蟲劑、突變劑、致癌劑等。結合反應的活性,可顯示該昆蟲是否具有抗藥的能力,同時可鑑定glutathione S-transferase的酵素次單位。

將野生型的果蠅培養於含有0.3%(v/v)phenol的培養基中發現,存活下來的果蠅所產生之子代相較於野生型親代有比較低的死亡率。經過GSH-S-Agarose affinity column純化得到的子代glutathione S-transferase酵素,利用1-chloro-2,4-dinitrobenzene作為受質(substrate),可偵測出比野生型親代更高的酵素活性。

將純化得到的子代glutathione S-transferase酵素,進行SDS-PAGE電泳分析發現處理一的F9子代與處理二的F8子代,比從野生型親代中純化的glutathione S-transferase酵素次單位多出一條帶(extra band),顯示經由0.3%(v/v) phenol刺激之後會改變glutathione S-transferase酵素的活性以及次單位的結構,同時也可以得知,glutathione S-transferase酵素可經由毒性物質的誘導,而增強其解毒活性。此點對於環境中有機毒物的解毒,是一個正面的訊息。
Abstrat
Glutathione S-transferases(GSH),which catalyze conjugation of reduced glutathione with various xenobiotics, including insecticides,mutagens,and carcinogens are cytosolic enzymes. By direct assays of GSH enzyme activity,we may detect potential drug resistance in insects. By analysis of SDS-polyacrylamide gel electrophoresis(SDS-PAGE),we may determine the multiple forms of subunits of an enzyme. The fruit flies(Drosophila melanogaster) were fed with medium containing 0.3% phenol five generations, lethality test, enzymatic assays for 1-chloro-2,4-nitrobenzene(CDNB) specific activities, as well as SDS-PAGE analysis were performed between untreated- and treated-flies.

Lethality teat revealed that the lethality rates of the offspring of treated-flies was significant lower than those of untreated flies, suggesting that phenol-resistant strain has been induced.The results of enzymatic assays indicated that the offspring of treated-flies has significantly higher enzymatic activities than those of untreated-flies using CDNB as a substrate.Besides,SDS-PAGE analysis of affinity-purified GST from untreated-flies and treated-flies(F5-F9)indicated that there was an extra band on the gel in F8 and F9.
As a summary, the results suggest that the phenol-resistant strain in D. melanogaster has been induced, and maintained. The capacity of resistance for phenol might be accounted for by higher CDNB activities, which was caused by the induction of an GST isozyme by phenol stimulation. Therefore, it is affirmative that upon stimulation of toxic substances, GST would be induced to increase its enzymatic activities in order to remove the toxic substances.
目錄
縮寫字對照表
中文摘要
英文摘要
緒論 1
實驗材料 4
實驗方法:
ㄧ.野生型果蠅的飼養 4
二.以化學藥物處理果蠅的飼養 4
三.野生型果蠅與以化學藥物處理果蠅的死亡率的比較 5
四.未純化蛋白質的取得 6
五.酵素的純化 6
六.蛋白質的分析 7
七.酵素活性的分析 7
八.蛋白質電泳分析 8
結果:
ㄧ.死亡率的比較 9
二.酵素活性的分析 12
三.蛋白質電泳分析 15
討論 17
附錄一 19
附錄二 19
附錄三 20
附錄四 22
附錄五 24
附錄六 27
附錄七 30
附錄八 31
參考文獻 32
縮寫字對照表

APS Ammonium persulfate
BSA Bovine serum albumin
CDNB 1-Chloro-2,4-dinitrobenzene
GST Glutathione S-transferase
GSH Redused glutathione
HOAc Acetic acid
MeOH Methanol
PTU Phenylthiourea
SDS Sodium dodecyl sulphate
TEMED N,N,N’-Tetraethylmethylene diamide
TRIS Tris(hydroxylmethyl)aminomethane
參考文獻

Baqlabaskaran S.,Chuen S.S. and Muniandy S. (1989) Glutathion S-transferase from the diamond back moth (Plutella xylostella Linnaeus). Insect Biochem. 19,453-443.

Booth J., Boyland E. and Smith P. An enzyme from rat liver catalyzing conjugation with glutathione. Biochem. J 79,516-524. (1961)

Cheng F. Y., Chou J.M. and Kao C.H. (1983) Inscticide resistance study in plutella xylostella (L.) IV. The activities of GST in the organophosphorous-resistant. J. Agric. Res. China. 32,373-378.

Chien, C. and Dauterman, W. C. (1991) Studies on glutathione S-transferase in Helicoverpa (=Heliothis) zea. Insect Biochem. 21,857-864.

Clark A.G., and Drake B. (1984) Purification and properties of glutathione S-transferase from larvae of Wisena cervinata. Biochem. J. 217,41-50.

Clark A.G., Dick L.G., Martindale M.S. and Smith N.J. (1985) Glutathione S-transferase from the new Zealand grass grub, Costelytra zealandica. Insect Biochem.15,35-44

Cochrane B.J. Morrissey J. and LeBalance, G. (1987) The genetics of xenobiotic metabolism and Drosophila-IV Purification and characterization of the major glutathione S-transferase. Insect Biochem. 17,731-738.

Cochrane B.J. and LeBlance, G. (1986) Genetics of xenobiotic metabolism in Drosophila I. Genetic and environmental factors affecting glutathione S-transferase in larvae. Biochem. Pharmac. 35,1679-1684

Dykstra W.D. and Dauterman W.D. (1978) Excretion, Distribution and Metabolism of S-(2,4-Dinitrophenyl) Glutathione in the American Cockroach. Insect Biochem. 8,263-265.

Fukami J. and Shishido T. (1966)Nature of a soluble glutathione-dependent enzyme system active a cleavage of methyl parathion to demethyl parathion. J. Econ. Entomol. 59,1338

Habig W. H., Pabst M. J., and Jacoby W.B. (1974) Glutathione S-transferase: The first enzymatic step in mercapturic acid formation,. J. Biol. Chem. 249,7130-7139

Haezelton G. A. and Lang C.A. (1983) Glutathione S-transferase activities in the yellow-fever mosquito [Aedes aegypti (Louisville)] during growth and aging. Biochem. J.210,281-287.

Hunaiti A.A.,Elbertieha M.A.,Owais M.W. (1995) Developmental Studies on Drosophila melanogaster Glutathione S-transferase and its Induction by Oxadiazolone. Insect Biochem. Molec. Biol. 25,1115-1119.

Jackoby W.B. (1978) The glutathione S-transferase: a group of multifunctional detoxication enzymes. Adv .Enzym. Relat. Areas molec. Biol.46,383-414.

Jansen M.,Baars J.A. and Breimer D.D. (1984) Cytosolic glutathione S-transferase in Drosophila melanogaster. Biochem. Pharmac. 33,3566-3659.

Mannervik B. (1985) The isoenzymes of glutathione transerase. Adv. Enzymol. 57,357-417.

Motoyama,N. and Dauterman W.C.(1977) Purification and properties of house fly glutathione S-transfersae. Insect Biochem. 7,361-369.

Toung Y.-P.S.,Hsieh T.-S. and Tu C.-P. (1990) Drosophila glutathione S-transferase region of sequence homology with the maize glutathione S-transferase III. Proc. Natn. Acad. Sci. U.S.A. 87,31-35
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