斑節蝦眼的蛋白質牻牛兒牻牛兒基轉移?I之純化與定性

Abstract

本論文是以水生無脊椎動物斑節蝦的眼為材料，藉以勝?受質KLKCFFL的酵素活性檢測方法，先後經過HiTrap?Q陰離子交換管柱層析，Superdex TM200膠體過瀘管柱層析，Mono Q陰離子交換管柱層析，最後以勝?偶合親合力管柱完成蛋白質牻牛兒牻牛兒基轉移?I (PGGT I)的純化。純化的PGGT I比活性為188 units/mg。從膠體過濾管柱層析估算的分子量為67 kDa與SDS-PAGE所估算的66 kDa相近，推測斑節蝦蛋白質牻牛兒牻牛兒基轉移?I為單體結構。所得的蛋白質被抗法呢基轉移?α次單元的抗體(Y-53)所辨認，顯示二者含有相似的胺基酸序列。 在所檢測的勝?中，KCFFL是較好的勝?受質。酵素作用最適的酸鹼值是pH 8.0。斑節蝦蛋白質牻牛兒牻牛兒基轉移?I證實是一種金屬性?（EDTA的I.C.50=0.25μM）但活性會受Mg2+（I.C.50=500μM）及Zn2+ (I.C.50=50μM）所抑制。根據各組織的酵素活性檢測，廣布於斑節蝦的眼、肝胰臟、心臟、鰓、神經節、腸、肌肉及殼各組織。

Protein geranylgeranyltransferase I was purified from the eyes of shrimp Penaeus japonicus by the sequentail column chromatography on HiTrap? Q, Superdex? 200, Mono Q and peptide-coupled affinity column. The purified enzyme was found to have relative Mr of 66 kDa as estimated by SDS-PAGE. Since the active protein geranylgeranyltransferase I from Superdex? 200 was found to have relative mass of 67 kDa, the purified enzyme was deduced to be a monomer. Synthetic peptide KCFFL is the best substrate of the protein geranylgeranyltransferase I among the investigated peptides. The optimal pH for enzyme activity is pH 8.0. The enzyme was inhibited by Mg++ and Zn++ ions at 50μM and 500μM respectively. Since EDTA (IC50=0.25μM) is inhibitory as well, the protein geranylgeranyltransferase I of Penaeus japonicus is a metaloenzyme.