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大白鼠小腸黏膜轉胺酵素的研究

大白鼠小腸黏膜轉胺酵素的研究.

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大白鼠小腸黏膜轉胺酵素的研究

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  1. 大白鼠小腸黏膜轉胺酵素的研究 中文摘要轉廣泛地存在於生物體中，它是一群在生化及免疫學上有相同特性的酵素，其可將蛋白質上的酸和離酸或多素以同鍵聯結，釋放出氨。這個反應需要鈣離子作為共同因子藉以活化轉，且此反應是發生在蛋白質轉譯後的調節。在人類的血液﹑皮膚﹑及各組織，轉也以不同的型態出現。最近我們發現一個新種類的轉，它存在於老鼠腸黏膜細胞，且不需要鈣離子來活化。我們以飽合硫酸銨沉澱，透析膜透析 (MW12,000)，膜過濾離心法濃縮蛋白質(Millipore biomax 10k)，DEAE陰離子交換色層分析 (BioRad)，凝膠過濾色層分析 (PharmaciaSephacryl S-在觀察溫度對轉的影響時，我們將轉加熱至37℃或50℃。不需鈣轉在37℃時，隨著加熱的時間增長其活性並無明顯的下降，但是需鈣轉其活性卻隨著加熱的時間增長呈現明顯的下降，可見不需鈣轉在37℃時比需鈣轉還要穩定。但加熱至50℃時需鈣與不需鈣轉都不穩定而失去活性。當加入蛋白抑制劑 (PMSF,leupeptin, Pepstatin) 於緩衝溶液中時，不需鈣轉活性明顯降低，需鈣轉只略為下降。我們推論此不需鈣轉是藉著蛋白的部份分解而活化。最後，作老鼠泡水至胸口數小時而後讓其復原的時間過程實驗，將老鼠泡水2﹐4﹐6小時，取出復原2﹐4﹐8, 12小時在不同的時間點做其十二指腸冷凍切片，再以免疫染色法測轉在十二指腸的位置和量的多寡及以去氧核醣核酸原位標定法來測十二指腸細胞自殺的程度和位置。結果發現隨著老鼠泡水時間增長，其十二指腸黏膜的轉量明顯提高，且因細胞自殺產生去氧核醣核酸斷裂的程度亦隨之增加。所以在老鼠十二指腸黏膜糜爛及復原過程中，轉之活化和細胞自殺的引發可能扮演著相當重要的角色。

  2. Study on rat intestinal mucosal transglutaminase • AbstractTransglutaminases (TGase) are a family of calcium-dependent enzyme catalyzing an acyltransfer reaction betweenpeptide bound glutamine and lysine residues or primary amine. Anovel calcium-independent TGase-like enzyme has beendemonstrated recently in mouse intestinal mucosal cells. Topurify this novel enzyme, saturated ammonium sulfate was used tofractionate the cytosol preparations. The majority of calcium-independent TGase activities was detected in the fractionprecipitated at 45%~55% saturated ammonThermal stability of thisnovel enzyme was studied at 37(C and 50(C. The calcium-independent TGase is more stable than calcium-dependenttransglutaminase at 37(C. However, activities of both enzymewere abolished at 50(C. When protease inhibitors ( PMSF,Pepstatin, Leupeptin ) were included in the homogenizing buffer,the enzyme activity was decreased as compared to that preparedin the abscense of protease inhibitors.The effect of stress andtissue repair on rat intestinal mucosal transglutaminase wasstudied by immunohistochemical method. The intensity of stressof transglutaminase-immunostaining increased in the intestinalmucosal tissue with the duration. To investigate whether stresscauses apoptosis in duodenal mucosal villus. DNA fragmentationinduced by apaptosis was assessed by in situ detection. Thephenomenon of apoptotic DNA fragmentation was observed in theintestinal mucosal villi. It is suggested that the

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