虎皮蛙阿爾法二巨球蛋白之純化及特性分析

1. 虎皮蛙阿爾法二巨球蛋白之純化及特性分析 • 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A2M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。

2. Purification and Characteriaztion of the alpha 2 Macroglobulin Protease Inhibitor from the Rana trigrin • The alpha 2 Macroglobulin (A2M) are classified as broad-spectrum inhibitorsbecause of their ability to entrap proteaseof different specificites and cata-lytic class. In the previousstudies from our laboratory, the putative frog A2McDNAcontaining thioester region sequences has been cloned. In thisreport, theA2M-like protease inhibitor from plasma of the Ranatrigrina was purified to a-pparent homogeneity by DEAE-sepharoseCL-6B anion-exchange column and preparat-ive electrophoresis.The purified protein resembled verterbrate A2Ms in thatitdisplays a broad specificity and inhibited the activity oftrypsin, chymotryps-in, papain, pepsin and elastase. Sensitivityof this purified protein to methy-lamine and autolytic cleavagesuggested the presence of an internal bata-cyste-inyl-r-glutamylthioester. The purified protein was tetramer of 180KDa and hadand 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 underwent autolytic fragmentation to produce two bandsofapproximately 110 and 80KDa resemble to human A2M. The aminoacid compositi-on showed similarities with the well-characterized vertebrate A2Ms. These resu-lts suggested thepurified protein was an A2M-like protease inhibitor.