Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase.
The effect of Mg2+ on the thermal inactivation and unfolding of rabbit muscle creatine kinase has been studied for various temperatures and Mg2+ concentrations. Increasing the Mg2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of creatine kinase during thermal denaturation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47 degrees C the increased free Mg2+ concentration caused the creatine kinase inactivation rate to increase. Increasing the temperature strengthened the effect of Mg2+ on the thermal inactivation. Control experiments showed that treating native creatine kinase with different concentrations of Mg2+ did not change the enzymatic activity. The fluorescence emission spectra showed that the emission maximum for creatine kinase red-shifted from 335 to 337 nm during thermal denaturation at 47 degrees C for 10 min, while the presence of 3 mM Mg2+ caused the enzyme emission maximum to red-shift from 335 to 342.5 nm for the same thermal denaturation conditions. In addition, Mg2+ also enhanced the unfolding of the equilibrium state and decreased the time required to reach the equilibrium state of creatine kinase at 47 degrees C. The potential biological significance of these results are discussed.
Cao
Z F
ZF
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, People's Republic of China.
Luo
W
W
Zhou
H M
HM
D000818:Animals; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D003402:Creatine Kinase; D008274:Magnesium