利用離子交換管柱與逆相高壓液相層析管柱，從台灣雨傘節分離出三個類三環結構之蛇毒， BM8，BM11及BM14。 BM8及BM14是具有82個胺基酸10個Cysteine residues的兩個蛇毒蛋白，它們最大的不同是在第37、 38個胺基酸(BM8是Glu37-Ala38；BM14是Lys37-Lys38)，而BM11則是具有68個胺基酸10個Cysteine residues的蛇毒蛋白。 但與Cardiotoxins及a-Neurotoxins相比其中八個Cysteine位在保留性位置，在N端另多了兩個Cysteine。 CD spectra顯示三者皆為b-sheet結構與a-Neurotoxins及Cardiotoxins相同。 BM8與BM14一級結構雖類似於Cardiotoxin卻無Cytotoxicity活性，而 BM14與BM11皆可有效抑制[3H]QNB與Muscarinic Acetylcholine receptor (M2)，結合但是BM8則僅有微弱的能力。 將BM14 Lysine residues進行化學修飾，則抑制作用下降，顯示Lys residues參與 BM14與 mAchR結合。 藉由PCR反應選殖BM14 genomic DNA的大小約為2.3 kb；BM11則約2.4 kb，其基因結構與Cardiotoxins、a-Neurotoxins 相同，具有3個Exons 2個Introns，顯示具有演化的同源性。 此特性亦表現在Promoter region上，數個已被證實存在的Transcription factor binding sites亦出現在BM11或BM14的Promoter region內，例如SP1、CACCC-factor binding site及NF-KB等。 另外，若與Cardiotoxins及a-Neurotoxins 基因相比較，在Intron部份大約有85%的相似性，至於Exon區域則有較高的變異性，此一結果顯示BM8、BM14及BM11與 a-Neurotoxins及Cardiotoxins在演化上應可歸類為同一基因家族，其變異性可能與適應性的演化相關。

Three three-finger proteins (BM8, BM11, BM14) were isolated from Bungarus multicinctus (Taiwan banded krait) venom using successive chromatography on a ion-exchanger column and reverse phase HPLC column. BM8 and BM14 contain 82 amino acid residues including 10 cysteine residues, and BM11 contain 68 amino acid residues including 10 cysteine residues. Unlike snake venom cardiotoxins and a-neurotoxins, the three proteins contain two additional cysteine residues in their N-terminal region. Noticeably, only two amino acid substitutions occurred at positions 37 and 38 were observed between BM8 and BM14. CD measurement revealed that their secondary structures were dominant with b-sheet structure as noted with cardiotoxins and a-neurotoxins. However, the gross conformations of BM8, BM11 and BM14 were not the same as evidenced by CD spectra and acrylamide quenching studies. In contract to BM8, BM11 and BM14 exhibit an activity on blocking [3H]QNB binding to muscarinic acetylcholine receptor (mAChR). Modification of Lys residues in BM14 resulted in a decreased binding activity, indicating that the Lys residues may be involved in the mAChR binding. The genomic DNA with the size of approximate 2.3 kb and 2.4 kb, respectively, encoded the precursors of BM14 and BM11 was amplified by PCR. The genes shared virtually identical overall organization with snake venom cardiotoxin andα-neurotoxin genes, composed of three exons and two introns. Comparison of BM11 and BM14 genes with cardiotoxins and a-neurotoxins genes showed that the exon regions were more diversified than intron regions. This implicated that the accelerated evolution may be involved in the evolution of these genes in order to acquire newly arising functions. Nevertheless, several transcriptional factor binding sites including SP-1, CACCC-binding factor and NF-KB were highly conserved in the promoter regions. It reflected a common regulation mechanism in controlling the transcription of these genes. These observations indicate that the three proteins are evolutionarily related to cardiotoxins and a-neurotoxins.

中文摘要 ------------------ 1
英文摘要 ------------------ 3
縮 寫 --------------------- 5
序 言 --------------------- 6
實驗材料 ------------------ 12
實驗方法 ------------------ 13
實驗結果 ------------------ 22
討 論 --------------------- 33
圖 表 --------------------- 38
參考文獻 ------------------ 67

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