臺灣博碩士論文加值系統

蘋果酸酶 (malic enzyme, ME)可催化氧化脫羧反應。在催化蘋果酸 (malate) 生成丙酮酸 (pyruvate) 和二氧化碳 (CO2)，同時將輔因子NAD(P)+還原成NAD(P)H。蘋果酸酶有三種異構型，分別是細胞質NADP-依賴型蘋果酸酶 (cytosolic NADP-dependent malic enzyme, c-NADP-ME, ME1)、人類粒線體NADP-依賴型蘋果酸酶 (mitochondrial NADP-dependent malic enzyme, m-NADP-ME, ME3)和人類粒線體NAD(P)+-依賴型蘋果酸酶 (mitochondrial NAD(P)+-dependent malic enzyme, m-NAD(P)-ME, ME2)，其中m-NAD(P)-ME能運用NAD+或NADP+兩種輔因子，較傾向於使用NAD+。相較於其他異構型，m-NAD(P)-ME在癌細胞中表現量高，能運用範圍較廣，研究價值較高。根據dbSNP和BioMuta資料庫，顯示了m-NAD(P)-ME之單一核苷酸多型性 (single-nucleotide polymorphisms, SNPs) 及癌症單一核苷酸變異 (single-nucleotide variations, SNVs)。我們試著以定點突變方式模擬這些突變型，且藉由酵素動力學試驗及四級結構的解析來分析這些酵素之特徵，包含酵素活性 (enzyme activity)、活化 (activation)、抑制 (inhibition) 及四級結構 (quaternary structure) 的改變，並比較野生型與突變型之間差異來了解這些突變對酵素之影響。藉由對m-NAD(P)-ME SNV/SNP的了解，希望未來m-NAD(P)-ME在預防及治療癌症上能提供多一種選擇。

Malic enzyme (ME) is an enzyme that catalyzes an oxidative decarboxylation reaction, which plays an important role in producing energy to maintain cell survival, and participates the major element production in the tricarboxylic acid cycle (TCA cycle). ME can fast catalyze malate to pyruvate and carbon dioxide (CO2), with conversion of NAD(P)+ to NAD(P)H at the same time. ME has three isoforms, including cytosolic NADP+-dependent malic enzyme (c-NADP-ME, ME1), mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME, ME2), and mitochondrial NADP+-dependent malic enzyme (m-NADP-ME, ME3). Therein, m-NAD(P)-ME can use either NAD+ or NADP+ as the cofactor, but prefers NAD+ under physiological conditions. Comparing to other isoforms, the m-NAD(P)-ME over-expressed in the cancer cell has a broader range of application and a higher value in research. The two databases, dbSNP and BioMuta, revealed the m-NAD(P)-ME associated single-nucleotide polymorphisms (SNPs) and the m-NAD(P)-ME associated single-nucleotide variations (SNVs) in various types of cancer. Hence, we try to simulate these SNP/SNV mutations by using site-directed mutagenesis, and analyze their enzyme characteristics by enzyme kinetic analysis, including enzyme activity, ATP inhibition, and fumarate activation. As well as we also analyze the protein quaternary structure of m-NAD(P)-ME by analytical ultracentrifuge (AUC). Furthermore, we can compare the difference between those mutations and wild-type to understand the influence of these SNPs and SNVs. It will be able to provide a way through m-NAD(P)-ME to inhibit or prevent cancer in the future.

摘要 i
Abstract ii
縮寫表 iv
表目錄 vi
圖目錄 vii
第一章、 前言 1
一、 蘋果酸酶 1
二、 單一核苷酸多型性和單一核苷酸變異 5
三、 雙羥萘酸 6
四、 研究目的 6
第二章、 實驗方法 8
一、 勝任細胞 (competent cell) 8
二、 定點突變 (site-directed mutagenesis) 8
三、 轉型作用 (transformation) 9
四、 抽取質體DNA (plasmid DNA extraction) 10
五、 誘導蛋白表現 (induction) 10
六、 蛋白質純化 11
七、 蛋白質品質分析 12
八、 酵素動力學分析 (enzyme kinetic assay) 14
九、 蛋白質四級結構分析 (analysis of protein quaternary structure) 18
第三章、 實驗結果 20
第四章、 討論 26
第五章、 結論 31
參考文獻 32
附錄 80
一、 實驗材料 81
二、 實驗器材與儀器 83
三、 藥品配製 85 

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