臺灣博碩士論文加值系統

人類醇脫氫酶(alcohol dehydrogenase；ADH)為複雜的酶族，有多個同功酶(isozyme)兼具有多種基質專一性。各種不同的一級和二級脂肪醇代謝中，ADH是其限速步驟。甲醇 (methanol)、乙二醇 (ethylene glycol)及異丙醇 (isopropanol)為臨床急診醫學最常遇到的三種工業毒醇，但與人類ADH酶族氧化代謝的關係所知甚少。當人誤食中毒時，在臨床所作急救處理，常以乙醇靜脈灌注或注射四甲基吡唑(4-methylpyrazole；4MP)合併血液透析來解毒。本研究探討人ADH族的乙醇和四甲基吡唑與工業常見毒醇包括甲醇、異丙醇、乙二醇的動力學交互作用。人類重組第一類ADH1A、ADH1B1、ADH1B2、ADH1B3、ADH1C1和ADH1C2與第二類ADH2及第四類ADH4對乙醇氧化的恆態動力學，與四甲基吡唑對重組ADH的抑制動力學機制，實驗在0.1 M NaPi(pH 7.5，25°C)含0.5 mM NAD+ (模擬肝細胞液輔酶濃度)的緩衝液中測定。 (1) ADH族中ADH1B1對甲醇氧化的Km為2.0 mM，其餘成員對甲醇氧化具較高Km (180–3500 mM)；Vmax範圍為0.025–0.41 U/mg。(2) ADH族中ADH1B1對乙二醇氧化的Km為4.3 mM，其餘成員對乙二醇氧化具較高Km (49–2600 mM)；Vmax為0.074–3.1 U/mg。(3) ADH族中ADH1A、ADH1B1、ADH1C1及ADH1C2對異丙醇氧化的Km分別為0.73、1.1、5.3及6.7 mM，其餘成員對異丙醇氧化具較高Km (160–3400 mM)；Vmax為0.045–3.7 U/mg。其中ADH2及ADH4對甲醇的氧化不具活性，而ADH1B3對乙二醇氧化亦不具活性。(4) 四甲基吡唑抑制ADH族乙醇氧化之酶動力學機制：四甲基吡唑抑制ADH1A、ADH1B1、ADH1B2、ADH1C1及ADH1C2氧化乙醇為競爭型；抑制ADH1B3、ADH2及ADH4氧化乙醇為非競爭型；斜率抑制常數(slope inhibition constants) Kis範圍在0.062–960 μM，截距抑制常數(intercept inhibition constants) Kii則在33–3000 μM。利用電腦模擬，將以上實驗測定之動力學參數帶入抑制方程式計算，預測毒醇在50 mM的高濃度情況，利用20 mM乙醇或是50 μM 四甲基吡唑皆可有效抑制毒醇的氧化。本研究結果從藥物遺傳學的觀點，為臨床使用四甲基吡唑或乙醇治療甲醇和乙二醇中毒患者，提供酶動力學的理論基礎依據。

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1 M sodium phosphate buffer, at pH 7.5 and 25◦C, containing 0.5 mM NAD+ and varied concentrations of substrate. Km values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12–57 mM, for methanol to be 2.0–3500 mM, for ethylene glycol to be 4.3–2600 mM, and for isopropanol to be 0.73–3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for Vmax for the toxic alcohols were much less than that of the Km across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (Kis) for the whole family being 0.062–960 μM and the intercept inhibition constants (Kii), 33–3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50 mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.

目錄 I
表目錄 III
圖目錄 IV
縮寫表 VI
中文摘要 VII
英文摘要 VIII
緒言 1
材料與方法 7
壹、實驗材料 7
一、化學藥品 7
二、主要儀器 8
三、載體與大腸桿菌宿主品系 8
貳、實驗方法 9
一、ADH活性之測定 9
二、人類重組醇脫氫酶族的誘導表現及純化 9
三、電泳 12
四、蛋白質濃度之測定 15
五、比活性之計算 15
六、醇脫氫酶動力學之分析 14
七、同時催化兩個不同基質之動力學分析 15
實驗結果 16
壹、醇脫氫酶族乙醇氧化之飽和動力學 16
貳、醇脫氫酶族毒性醇氧化之動力學 17
一、甲醇氧化之動力學分析 17
二、乙二醇氧化之動力學分析 17
三、異丙醇氧化之動力學分析 17
参、醇脫氫酶族乙醇氧化之死巷抑制實驗 18
一、人類重組第一類醇脫氫酶ADH1A 18
二、人類重組第一類醇脫氫酶ADH1B1 18
三、人類重組第一類醇脫氫酶ADH1B2 18
四、人類重組第一類醇脫氫酶ADH1B3 18
五、人類重組第一類醇脫氫酶ADH1C1 18
六、人類重組第一類醇脫氫酶ADH1C2 18
七、人類重組第二類醇脫氫酶ADH2 19
八、人類重組第四類醇脫氫酶ADH4 19
肆、模擬乙醇及四甲基吡唑抑制毒醇之氧化比率 19
一、乙醇抑制ADH酶族對毒醇氧化抑制率之模擬 19
二、四甲基吡唑抑制ADH酶族對乙醇及毒醇氧化抑制率之模擬 19
討論 21
壹、人類醇脫氫酶族之乙醇與毒性醇之交互作用 21
貳、臨床上毒醇中毒與治療之應用 22
結論 23
參考文獻 24








 
表目錄
頁碼
表一 人類 ADH 族之生物及結構特性 28
表二 人類 ADH 族基質及輔酶結合部位之重要胺基酸 29
表三 人類不同族群ADH對偶基因之分佈頻數 30
表四 重組人類ADH族對乙醇氧化之動力學常數 31
表五 重組人類ADH族對甲醇氧化之動力學常數 32
表六 重組人類ADH族對乙二醇氧化之動力學常數 33
表七 重組人類ADH族對異丙醇氧化之動力學常數 34
表八 重組人類ADH族對乙醇及毒性醇Vmax (U/mg) 之比較 35
表九 重組人類ADH族對乙醇及毒性醇Vmax/KM (mU/(mg mM))之比較 36
表十 4-methylpyrazole對重組人類ADH族催化乙醇氧化之抑制型和抑制常數 37
表十一 計算機量化模擬4-methylpyrazole抑制ADH族之乙醇氧化 38



 
圖目錄
頁碼
圖一 人類ADH族重組蛋白之純化流程 39
圖二 人類重組蛋白ADH1A催化甲醇、乙二醇、異丙醇及乙醇之氧化飽和曲線圖 40
圖三 人類重組蛋白ADH1B1、ADH1B2與ADH1B3催化甲醇、乙二醇、異丙醇及乙醇之氧化飽和曲線圖 41
圖四 人類重組蛋白ADH1C1與ADH1C2催化甲醇、乙二醇、異丙醇及乙醇之氧化飽和曲線圖 42
圖五 人類重組蛋白ADH2與ADH4催化甲醇、乙二醇、異丙醇及乙醇之氧化飽和曲線圖 42
圖六 人類重組蛋白ADH1A乙醇氧化之4-methylpyrazole死巷抑制. 44
圖七 人類重組蛋白ADH1B1、ADH1B2與ADH1B3乙醇氧化之4-methylpyrazole死巷抑制 45
圖八 人類重組蛋白ADH1C1與ADH1C2乙醇氧化之4-methylpyrazole死巷抑制. 46
圖九 人類重組蛋白ADH2與ADH4乙醇氧化之4-methylpyrazole死巷抑制. 47

圖十 量化模擬細胞內ADH1A重組蛋白之乙醇與4-methylpyrazole交互作用. 48
圖十一 量化模擬ADH1B1、ADH1B2與ADH1B3重組蛋白之乙醇與4-methylpyrazole交互作用 49
圖十二 量化模擬ADH1C1與ADH1C2重組蛋白之乙醇與4-methylpyrazole交互作用. 50
圖十三 量化模擬ADH2與ADH4重組蛋白之乙醇與4-methylpyrazole交互作用. 51
圖十四 量化模擬人類ADH重組蛋白之乙醇對methanol、ethylene glycol以及isopropanol氧化抑制百分比. 52
圖十五 量化模擬人類ADH重組蛋白之4-methylpyrazole對 ethanol、methanol、ethylene glycol以及isopropanol氧化抑制百分比. 53

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