| Summary: | 碩士 === 台北醫學院 === 細胞及分子生物研究所 === 87 === 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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