蔡蔭和臺灣大學：生化科學研究所廖聰榮2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52823藉由三段的純化程序，我們從印尼金黃樹蝮 (Trimeresurus puniceus) 蛇毒中純化出4個蛇毒絲胺酸蛋白酶Tpu1—4。以三種呈色基質 (chromogenic substrate)測試顯示，Benzoyl-Pro-Phe-Arg-p-NA是Tpu1較好的基質，而Tosyl-Gly-Pro-Arg- p-NA對於Tpu2、Tpu3是較好的基質，但Tpu4對這三種基質皆無明顯反應。並且也發現Tpu1—4皆會切割高分子激肽原（high molecular weight kininogen, HMWK）、血管收縮素Ⅰ (angiotensinⅠ) 與血纖維原 (fibrinogen)。利用SDS-PAGE觀測Tpu1—4分子量，經PNGase F (一種N-glycanase)處理作用後，Tpu1—4的分子量皆約為25~27kDa。而從蛇毒中純化之Tpu1和Tpu4比Tpu2和Tpu3具有較大的分子量，顯示Tpu1與Tpu4具有較高的醣化程度。另外，我們也用定序儀解出Tpu1—4各自的N端胺基酸序列，發現這些序列確實明顯不同。 我們使用適當的引子 (primers) 放大並選殖了印尼金黃樹蝮的蛇毒絲胺酸蛋白酶的互補核酸 (cDNA)，在分析了40個clones之序列後，我們得到三個較可信的互補核酸序列。由此核酸序列所推定出的胺基酸序列，分別由234、233和236個胺基酸所組成。依照這三個胺基酸序列的N端與純化的Tpu1—4的對應情形，分別確定即可能為Tpu1、Tpu2和Tpu4，其預估分子量分別為25624、25350和 26109 Da (假設皆有六對雙硫鍵的情況之下)，其活性中樞也皆有His57，Asp102，Ser195 (chymotrypsin numbering) 等對應成分。不同的是，Tpu1具有5個potential N-glycosylation sites，Tpu4具有4個，但Tpu2只有2個。 同樣我們也選殖了外觀相近之蛇種婆羅州黃樹蝮 (Trimeresurus borneenesis)的三個蛇毒絲胺酸蛋白酶的互補核酸，從核酸定序推定三個的胺基酸序列，分別將其命名為Tbo2、Tbo3和Tbo4，分別由233、234和236個胺基酸所組成，預估分子量分別為25295、25522和26149 Da (假設各絲胺酸蛋白酶分子皆有六對雙硫鍵) ， 它們分別具有2、2、4個potential N-glycosylation sites。有趣的是，我們發現Tpu3的N端胺基酸序列與Tbo3相同，而Tpu2與Tbo2只有三個胺基酸不同且都具有13個半胱胺酸 (cysteines)，這也許可以更確認這兩種樹蝮是相近的蛇類。 過去本實驗室也報告了許多由Trimeresurus stejnegeri所選殖出的絲胺酸蛋白酶的互補核酸序列及推定的胺基酸序列，經過序列比對後發現多數屬於KN (kininogenase)。利用BLASTP做序列搜尋及分析也發現本實驗所報告的序列與其他實驗室所報告來自台灣與中國Trimeresurus stejnegeri的蛇毒KN非常相似，用種屬樹 (phylogenetic tree) 的分析結果再次證實這三種同屬的樹蝮絲胺酸蛋白酶序列十分相近，這三種樹蝮主要表現有多種KN。Four serine proteases (designated Tpu1—4) were purified from the venom of Trimeresurus puniceus (golden tree viper) from Indonesia. Upon the amidolytic assays using three chromogenic substrates, Tpu1 preferred to hydrolyze Benzoyl-Pro-Phe-Arg- p-NA, a chromogenic substrate for kallilrein, Tpu2 and Tpu3 preferred to hydrolyze Tosyl-Gly-Pro-Arg-p-NA, a chromogenic substrate for thrombin, but Tpu4 did not hydrolyzed any of these substrates. These purified proteases were able to cleave high molecular weight kininogen (HMWK), fibrinogen, and angiotensinⅠ, but with different efficacies. The estimated molecular weights of Tpu1—4 under reducing condition (with 5% β-mercaptoethanol) were 54 kDa, 30 kDa, 35 kDa, 51 kDa, respectively. After removal of N-glycans by PNGaseF the reduced Tpu1—4 migrated as single bands with an apparent molecular weight of 25~27 kDa in SDS-PAGE. Tpu1 and Tpu4 apparently have higher carbohydrate content than Tpu2 and Tpu3. The N-terminal sequences of these purified proteases are different from each other. PCR of the venom gland cDNA mixtures using designed specific primers resulted in the amplification of cDNAs encoding these serine proteases. After checking the sequences of about 40 clones, three distinct cDNA sequences were found to match the proteases purified. The predicted molecular weight without post-translation modification (but assuming the formation of six pairs of disulfide bonds) for Tpu1, Tpu2 and Tpu4 are 25624 Da, 25350 Da, and 26109 Da, respectively. The active site residues of serine proteases family, i.e., His57, Asp102 and Ser195 (the chymotrypsin numbering) and location of six pairs of disulfide bonds of the viperid venom proteases are conserved. Tpu1, Tpu2 and Tpu4 contain five, two, and four potentional N-glycosylation sites, respectively. In addition, three distinct cDNA sequences encoding serine proteases were cloned from the venom gland of T. borneenesis, a species closely related to T. puniceus. The deduced proteases were named Tbo2, Tbo3 and Tbo4, respectively. Interestingly, the N-terminal sequence of Tpu3 is found to be identical to that of Tbo3. Tbo2 and Tpu2 differ by only three residues, confirming a very close relationship between T. puniceus and T. borneenesis. Results of BLASTP search and phylogenetic analyses also revealed that these serine protease sequences of T. puniceus and T. borneenesis are very similar to those of Chinese green tree viper Trimeresurus stejnegeri.目錄 中文摘要．．．．．．．．．．．．．．．．．．．．．．．１ 英文摘要．．．．．．．．．．．．．．．．．．．．．．．３ 縮寫字表．．．．．．．．．．．．．．．．．．．．．．．５ 材料與藥品．．．．．．．．．．．．．．．．．．．．．．６ 儀器設備．．．．．．．．．．．．．．．．．．．．．．．８ 緒論．．．．．．．．．．．．．．．．．．．．．．．．．９ 實驗方法．．．．．．．．．．．．．．．．．．．．．．１２ 實驗結果．．．．．．．．．．．．．．．．．．．．．．１７ 討論．．．．．．．．．．．．．．．．．．．．．．．．２２ 圖表及說明．．．．．．．．．．．．．．．．．．．．．２６ 參考文獻．．．．．．．．．．．．．．．．．．．．．．４４ 附錄．．．．．．．．．．．．．．．．．．．．．．．．４９913191 bytesapplication/pdfen-US樹蝮蛇毒絲胺酸蛋白酶serine proteasesTrimeresurus puniceusTrimeresurus borneenesisotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52823/1/ntu-94-R92b46033-1.pdf