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
中文摘要
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。
英文摘要
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.
