百步蛇類凝血酵素的分子構造及基質特擇性

Abstract

類凝血酵素（Thrombin-like enzyme簡稱TLE）是從管牙科毒蛇之蛇毒成分，試管中具有促凝的作用，並能水解fibrinogen，故具有抗血栓抗中風療效。利用DEAE Trisacryl(M）陰離子交換柱及TSK G2000SW HPLC過濾柱，我們從台灣百步蛇中，純化一個amidase活性最高，會促凝，而且會水解fibinoge A（?）鏈的TLE, 稱之為DATLE（即來自Deinagkistrodonacutus的TLE) ，它是一個醣蛋白，分子量據SDS-PAGE估計為41kDa ，經glycsidase將其N-linked giycans切除後，其分子量轉變成36kDa 。另以呈色基質研究DATLE、ancrod、crotalase三者的specificity constants，發現Tosyl-Gly-Pro-Arg-p-Nitroanilide是DATLE較好的基質，crotalase和 ancrod則對不同的基質有更好之特擇性。此外，我們已將DATLE的氨基酸序列解出約80%，並和其他TLE序列比對。 另外，經由DEAE Trisacryl(M）的分離純化，也發現台灣百步蛇毒中具有多種amidases，其活性不同，但分子量均在40?42kDa之間。以C4RP-HPLC純化後做N端氨基酸序列，結果其N端氨基酸序列大約20個殘基左右相同，均為TLE之N端序列。 在cDNA方面，利用蛇毒serine protease共通的PCR引子，找到了二種trypsin-like enzyme的cDNA，但皆與DATLE序列不相同，我們續用已知DATLE氨基酸序列設計引子，將用PCR等方法，繼續完成DATLE的基因選殖，以期得到其完整cDNA序列。 總之，在這些研究中，我們分析了百步蛇毒中amidase活性最高的TLE之分子構造及基質特擇性，並且發現TLE有其他isoforms並存，十分值得再加研究。

Thrombin-like enzymes(TLE) purified from crotalidae snake venoms are procoagulant in vitro and catalyze the hydrolysis of fibrinogen. Some of them have been used for the prevention and treatment of thrombotic disorders. ATLE (named DATLE) was purified from Deinagkistrodonacutus venom by DEAE Trisacryl(M) column and TSK G2000SW gel filtration HPLC, and showed strong amidase activity and fibrinolytic activity. Based on the results of SDSPAGE, its molecular mass was about 41kDa.After deglycosylaton by N-glycosidase, it reduces to 36kDa. We compared their specificity constants of DATLE, ancrod, and crotalase toward three kinds of chromogenic substrates. We found that Tosyl-G-P-R-pNA is the best substrate for DATLE, and that is different from the other two enzymes. About 80% of the amino acid sequence of DATLE was determined by protein fragmentation and automated sequencing. More than six amidases in the venom was found. These amidases are different in pI, amidase activity, but appeared to have the same TLEN-terminal sequences upto the 20th residue. By cDNA cloning and sequencing, we solved the nucleotide sequences of two trypsin -like proteins from the cDNA prepared form mRNA of the venom gland. But their deduced amino acid sequences are different from that of DATLE. Apparently, there are several variants of TLE in the gland. In summary, we have characterized and studied the kinetic specificity of the TLE. We also discovered its isoforms from D.acutus venom and variant cDNAfrom the venom gland. The structure-activity relationship of these TLE isoforms or variants remains to be studied.