Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase

Abstract

The e ect of Mg 2+ on the thermal inactivation and unfolding of rabbit muscle creatine kinase has been studied for various temperatures and Mg 2+ concentrations. Increasing the Mg 2+ concentration in the denatured system signi®cantly 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 478C the increased free Mg 2+ concentration caused the creatine kinase inactivation rate to increase. Increasing the temperature strengthened the e ect of Mg 2+ on the thermal inactivation. Control experiments showed that treating native creatine kinase with di erent concentrations of Mg 2+ did not change the enzymatic activity. The¯uorescence emission spectra showed that the emission maximum for creatine kinase red-shifted from 335 to 337 nm during thermal denaturation at 478C for 10 min, while the presence of 3 mM Mg 2+ caused the enzyme emission maximum to red-shift from 335 to 342.5 nm for the same thermal denaturation conditions. In addition, Mg 2+ also enhanced the unfolding of the equilibrium state and decreased the time required to reach the equilibrium state of creatine kinase at 478C. The potential biological signi®cance of these results are discussed. #

Effect of magnesium ions on the thermal stability of human poly(A)‐specific ribonuclease

“…Unfortunately, no metal ions are present in the crystal structure of the catalytic domain of PARN [17], which limits the understanding of the impact of the two ions on the catalysis as well as the structural stability of PARN. Magnesium, the second most abundant intracellular cation and the fourth most abundant cation in the body, has a well‐established influence on intracellular biological processes [24] and the functions, stability and folding of proteins as well as RNAs (for example [25–31]). The total magnesium content in various cell types ranges from 5 to 30 mM, while the concentration of the free Mg 2+ has been determined to be in the range of 0.3–3 mM [24,32].…”

Thermal Inactivation and Unfolding of a Dimeric Arginine Kinase

“…Extensive studies have been carried out on CK and monomer AK. The unfolding/refolding of CK in either denaturant solutions [4][5][6][7][8] or thermal denaturation [9,10] were reported previously, as was data for the monomer of AK [11,12]. However, in contrast, little research has been carried out on dimeric AK.…”

“…Since this AK is a dimeric enzyme with two subunits, it is important to explore the dissociation course of the enzyme during thermal denaturation. It has been established that if denaturation of oligomeric proteins includes a step of reversible dissociation to monomers, the apparent heat capacity change (∆C p ) vs. temperature profile becomes dependent on protein concentration, owing to the bimolecular character of association [10]. Therefore, we chose to investigate the thermal denaturation of this dimeric AK by DSC at three different concentrations.…”