- 學位論文
虎皮蛙阿爾法二巨球蛋白之純化及特性分析
Purification and Characteriaztion of the alpha 2 Macroglobulin Protease Inhibitor from the Rana trigrin
摘要
阿爾法二巨球蛋白(以下稱A2M)是一種高分子量並具有廣效性的蛋白 質水解酵素抑制分子(protease inhibitor)，因為A2M它能夠利用陷阱機 制來抑制體內多類系蛋白水解酵素。早先本實驗室已經選殖出虎皮蛙A2M 中硫酯位的互補去氧核糖核酸序列。在本研究中，以台灣地區所購玩o的 虎皮蛙之血清為材料，利用DEAE瓊酯糖CL-6B的陰離子交換樹脂及製備型 電泳法純化分離出均質的擬似A2M蛋白質水解酵素抑制分子。此純化出之 蛋白，經分析其特性非常類似脊椎動物的A2M分子，其具有相同廣效蛋白 質水解酵素之抑制性，此外經甲基銨處理測試及熱裂解檢測後，顯示分子 內部具bata-半胱氨醯-r穀銨醯銨醯硫酯位(internalbata-cysteinyl-r- glutamyl thioester)。將所純化出來之蛋白質，利用還原型及非還原型 之聚丙烯醯胺凝膠電泳確認其分子量約為720KDa之四合體化合物其單體分 子量約為180KDa。當純化出之蛋白質在含有硫酸十二酯鈉(SDS)中煮沸後 ，它會產生斷裂且在電泳圖上會產生約110及80KDa單體裂片，與人類的A2 M分子相似。此蛋白分子之胺基酸組成分析亦呈現與脊椎動物的A2M有許多 相似的特徵。綜合上述結果，顯示此純化出之蛋白分子為擬A2M蛋白質水 解酵素抑制分子。
並列摘要
The alpha 2 Macroglobulin (A2M) are classified as broad- spectrum inhibitorsbecause of their ability to entrap protease of different specificites and cata-lytic class. In the previous studies from our laboratory, the putative frog A2McDNA containing thioester region sequences has been cloned. In this report, theA2M-like protease inhibitor from plasma of the Rana trigrina was purified to a-pparent homogeneity by DEAE-sepharose CL-6B anion-exchange column and preparat-ive electrophoresis. The purified protein resembled verterbrate A2Ms in that itdisplays a broad specificity and inhibited the activity of trypsin, chymotryps-in, papain, pepsin and elastase. Sensitivity of this purified protein to methy-lamine and autolytic cleavage suggested the presence of an internal bata-cyste-inyl-r-glutamyl thioester. The purified protein was tetramer of 180KDa and had and apparent mass of approximately 720Kda when examined by non- reducing and re-ducing sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE), and gel filteration. When boiled in SDS it under went autolytic fragmentation to produce two bandsof approximately 110 and 80KDa resemble to human A2M. The amino acid compositi-on showed similarities with the well- characterized vertebrate A2Ms. These resu-lts suggested the purified protein was an A2M-like protease inhibitor.