Effect of Mg2+ during reactivation and refolding of guanidine hydrochloride-denatured creatine kinase.
Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296-1302; Yao et al. (1984), Biochemistry 23, 2740-2744; Yao et al. (1982), Sci. Sin. 25B, 1186-1193]. Under suitable conditions, about 60-70% of the activity can be recovered in the presence of different Mg2+ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg2+ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg2+; however, the rates of reactivation are independent of the Mg2+ concentration. In addition, Mg2+ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg2+ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg2+ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.
Park
Y D
YD
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China.
Zhou
H M
HM
D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D009132:Muscles / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D003402:Creatine Kinase; D008274:Magnesium; D019791:Guanidine