Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions.
It is well known that Mg(2+) is an essential component in many biological processes. This research investigated the courses of both the reactivation and the refolding in the absence and presence of Mg(2+) ions. Calf intestinal alkaline phosphatase (CIP) was extensively denatured in 3 M guanidine hydrochloride (GdnHCl) solution for 2 h. Under suitable renaturation conditions, about 60-70% of the activity was recovered in the absence and presence of different magnesium ion concentrations. The refolding processes followed two-phase courses, whereas the reactivation processes were monophasic after dilution in proper solutions with or without Mg(2+). The magnesium ions affected both the reactivation and the refolding courses of unfolded CIP. A comparison of rate constants for the refolding of unfolded CIP with those for recovery of enzyme activity at different Mg(2+) concentrations showed that they were not synchronized. The activity recovery was speeded up due to the presence of Mg(2+) ions; while the refolding course of unfolded CIP was somewhat inhibited by the excess Mg(2+).
Zhang
Ying-Xia
YX
Department of Chemistry, Capital University of Medical Science, Beijing 100054, PR China.
Zhu
Ying
Y
Xi
Hong-Wei
HW
Liu
Yong-Li
YL
Zhou
Hai-Meng
HM
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000378:metabolism; D058851:GPI-Linked Proteins; D019791:Guanidine / Q000378:metabolism; D066298:In Vitro Techniques; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000951:Antigens, Neoplasm; D004793:Enzyme Reactivators; D058851:GPI-Linked Proteins; C063088:ALPI protein, human; D000469:Alkaline Phosphatase; D008274:Magnesium; D019791:Guanidine