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大白鼠睪丸轉麩胺的純化與定性

大白鼠睪丸轉麩胺的純化與定性.

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大白鼠睪丸轉麩胺的純化與定性

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  1. 大白鼠睪丸轉麩胺的純化與定性 • 轉麩胺(Transglutaminase, TGase)隸屬於一類需要鈣離子活化的酵素群，它的催化作用在於醯基轉換(acyl-transfer)的反應，當它被鈣離子活化後，便可將一特定酵化物(substrate)的麩胺酸殘基(glutamine residue)與另一酵化物的離胺酸殘基(lysine residue)或多胺(polyamine)以共價鍵結合，這種鍵結穩定而且能抗化學物質、蛋白的侵蝕。這類酵素廣泛分佈於組織中，且參與了許多細胞內的活動，如生長、分化、增生、細胞自殺等。近年來發現轉麩胺中的組織轉麩胺(tissue TGase, type II TGase)具有結合、水解三磷酸鳥糞核糖(GTP)的能力，是一種新型態的G-蛋白質(G-protein)，稱做Gah。為了研究生殖系統中的轉麩胺，本實驗純化了Wistar氏大白鼠的睪丸轉麩胺。整個純化過程經數種不同的純化流程與方法之測試後，最後以DEAE離子交換管柱、Sephacryl S-200及GTP-agarose親和性層析管柱層析法的方式可完全純化出睪丸轉麩胺。這個方法可以使蛋白質達到約七千倍的純化及約14% 的回收率。純化出的蛋白質在SDS-電泳膠上以銀染色法處理顯示出一條分子量大約為77,000道耳吞(Dalton)的單一色帶。使用抗肝細胞組織轉麩胺單株一級抗體作西方點墨法分析，亦驗證這是組織轉麩胺。此酵素對其酵化物「N'N'-dimethylcasein」的反應飽和濃度為1 mg/ml；對另一酵化物「putrescine」的反應飽和濃度為1 mM，Km值約為0.035 mM。抑制劑的測試發現它的活性受到GTP的抑制遠大於ATP，也受到氯化鈉的輕微抑制；結構相似的一級胺測試則顯示此酵素傾向於與兩端不對稱的一級胺反應。另外此酵素亦具有水解GTP的能力。這份研究報告顯示存在於睪丸內的轉麩胺所表現出的特質應類屬於第二型轉麩胺，即「組織轉麩胺」。

  2. Purification and Characterization of Rat Testicular Transglutaminase • Transglutaminases (TGase) are a family of Ca++-dependent enzymes that catalyze an acyl-transfer reaction between peptidyl glutamine residues and primary amines, including the e-amino group of lysine residues in protein. The enzymes are widely distributed in tissues and have been reported to par-ticipate in many cellular processes, including growth and differentiation. Tissue transglutaminase are recently identified as a GTP-binding protein, Gah. In order to characterize testicular transglutaminase, several purification proto-cols were tested, and finally the Wistar rat testicular TGase was purified by a novel procedures including DEAE ion-exchange column, Sephacryl S-200 gel-filtration, and GTP-agarose affinity chromatographies. The method gave about 7,200-fold purification with a reproducible yield of about 14%. The purified enzyme showed as a 77 kDa single band on SDS-PAGE. Western blot analysis using anti-liver transglutaminase monoclonal antibody also identified the enzyme as tissue transglutaminase. Saturation reaction of N'N-dimethylcasein was 1 mg/ml, and that of putrescine was 1 mM for the enzyme. The Km value of putrescine for the enzyme was 0.035 mM. The enzyme inhibitor test showed that TGase activity was strongly inhibited by GTP rather than ATP, and was slightly inhibited by NaCl. The competitive inhibitory experiments with various substrate analogues demonstrated that testicular TGase preferred unsymmetrical primary amines to symmetrical ones. In addition, the enzyme also has GTP hydrolysis capacity. These data indicated that testicular TGase displays the properties of type II trans-glutaminase, the "tissue-type transglutaminase", as well as the Gah characteristics.

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