镁离子对碱性磷酸酶折叠过程的影响

对小牛肠碱性磷酸酶再折叠的机制及其影响因素进行了探讨。在 2 5℃室温下 ,以 3mol/L盐酸胍对小牛肠碱性磷酸酶 (CIP)进行了 2h的去折叠实验 ,然后再以Tris HCl缓冲液对变性体系进行 2 0倍稀释 ,观察该酶活力恢复的动力学过程并与镁离子参与下的稀释复性过程进行比较。并就不同实验条件下含镁稀释或不含镁稀释 ,获得的最终酶活力、酶活力恢复水平、活力恢复速率常数及数据的相对标准偏差等指标进行了探讨。认为镁是CIP位点的特异性“效应分子” ,它与酶中有关基团的配位 ,稳定了酶活性部位的构象 ,使得自发折叠过程能够向类天然态的方向进行

Effects of Magnesium Ions on Refolding of Calf Intestinal Alkaline Phosphatase

Recently the refolding studies have been concentrated on alkaline phosphatase from E. Coli. The present experiments studied the refolding of mammalian enzyme. Calf intestinal alkaline phosphatase (CIP) was denatured in 3 mol/Lguanidine hydrochloride solutions for 2 h at 25 ℃. Under suitable renaturation conditions, the kinetic courses of enzymatic activity recovery were investigated in the absence and presence of magnesium ions at 2 mmol/Lconcentration, and compared to each other. The two remarkably different courses characterized by the final values of activity recovery, recovery levels, kinetic constants of activity recovery and relative standard deviation of the data indicated that magnesium ions behaved like "site-specific" effectors which were bound to the concerning residues of enzyme protein and stabilized the conformation of active site domain, thus guided the refolding process in a productive pathway.