Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60 degrees C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A(lim)) is proportional to protein concentration at both temperatures. The first order rate constant (k(I)) does not depend on protein concentration in the range 0.05-0.2 mg/ml at temperature 50.6 degrees C, but at temperature 60 degrees C it increases with the growth of protein concentration in the range 0.1-0.4 mg/ml. Kinetic curves, shown in coordinates {A/A(lim); t}, in experiments at 50.6 degrees C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60 degrees C, half-transformation time t(1/2) = ln2/k(I) decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60 degrees C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6 degrees C aggregation rate is limited by the stage of protein molecule denaturation, but at 60 degrees C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation.

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The quantitative aspects of mitochondrial creatine kinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (Ka = 0.39 mM at ionic strength I = 7.5 mM and 5 degrees C); after protonation its affinity increased many times (Kah = 39 nM). Determination of mitCK adsorption capacity by another method at pH 7.4, when the enzyme is almost protonated, gave Kah = 15 nM. The effect of ionic strength on mitCK adsorption may be described in terms of Debye-Hückel's theory for activity coefficients assuming the charges of the interacting species to be +4 and -4. The dissociation constant for the mitCK-membrane complex at pH 7.4 and I = 0 was evaluated by different approaches as approx. 1 nM. Extramitochondrial ATP (or ADP) shifted greatly the equilibrium between the adsorbed and the free mitCK towards the solubilized state, since in the adsorbed protein the external ligands had access to four binding sites and in the free protein to eight sites.

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The kinetics of chemical modification of arginine residues in mitochondrial creatine kinase (mit-CK) from beef heart by 4-hydroxy-3-nitrophenylglyoxal (HNPG) have been studied with simultaneous registration of enzyme inactivation. Experiments showed that complete inactivation of mit-CK corresponded to modification of two arginine residues per mit-CK monomer. The data on the modification kinetics can be described by the sum of two exponential terms and suggest strong negative cooperativity in the binding of HNPG to arginine residues. The rate constants for the fast and slow phases of modification differ by a factor of about 50. The corresponding rate constants for inactivation differ by a factor of about 30. The rate constant for the slow stage of inactivation is twice as large as that for the rate constant for the slow stage of modification, i.e., the inactivation process is ahead of the modification process.