Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions

2002 ◽  
Vol 80 (4) ◽  
pp. 427-434 ◽  
Author(s):  
Wen-bin Ou ◽  
Ri-Sheng Wang ◽  
Hai-Meng Zhou
Keyword(s):  
Creatine Kinase ◽  
Dimethyl Sulfoxide ◽  
Conformational Changes ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Hydrophobic Surfaces ◽  
Muscle Creatine Kinase ◽  
Organic Mixture ◽  
Dmso Concentration ◽  
Partial Unfolding

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (<65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water–organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.Key words: creatine kinase, dimethyl sulfoxide, denaturation, activity, conformation.

scholarly journals Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase

1973 ◽  
Vol 135 (2) ◽  
pp. 265-276 ◽  
Author(s):  
B. Focant ◽  
D. C. Watts
Keyword(s):  
Creatine Kinase ◽  
Smooth Muscle ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Intermediate Stage ◽  
Hill Coefficient ◽  
Rabbit Muscle ◽  
Difference Spectra ◽  
Muscle Creatine Kinase ◽  
Quaternary Complex

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO3- or Cl- ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by αβ-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO3- ions to the enzyme–creatine–MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme–creatine–NO3-–MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO3- is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme–pyruvate–NO3-–MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or α-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K+-free enzyme. Only the former inhibits the K+-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.

scholarly journals Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K

1977 ◽  
Vol 167 (3) ◽  
pp. 731-737 ◽  
Author(s):  
J Williamson ◽  
J Greene ◽  
S Chérif ◽  
E J Milner-White
Keyword(s):  
Amino Acids ◽  
Creatine Kinase ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteinase K ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Muscle Creatine

By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3—MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.

scholarly journals A study of the role of the reactive thiol group of rabbit muscle creatine kinase with a chromophoric reporter group

1978 ◽  
Vol 171 (1) ◽  
pp. 269-272 ◽  
Author(s):  
M A Keighren ◽  
N C Price
Keyword(s):  
Creatine Kinase ◽  
Conformational Changes ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Transition State Analogue ◽  
Muscle Creatine ◽  
Reporter Group

Substrate- and ligand-induced conformational changes were studied in a series of thiol-modified derivatives of rabbit muscle creatine kinase that retained different amounts of enzymic activity. The results indicate that the ‘reactive’ thiol group of the enzyme is required for the conformational changes associated with formation of a ‘transition-state analogue’ complex.

Sodium barbital is a slow reversible inactivator of rabbit-muscle creatine kinase

2006 ◽  
Vol 84 (2) ◽  
pp. 142-147
Author(s):  
Feng Shi ◽  
Tong-Jin Zhao ◽  
Hua-Wei He ◽  
Jie Li ◽  
Xian-Gang Zeng ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Energy Homeostasis ◽  
Inactivation Kinetics ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Reversible Inactivation ◽  
Inhibition Effect ◽  
Sodium Barbital ◽  
Muscle Creatine ◽  
Detailed Kinetics

As a depressant of the central nervous system, the clinical effect of sodium barbital has been extensively studied. Here we report on sodium barbital as an inhibitor of rabbit-muscle creatine kinase (CK), which plays a significant role in energy homeostasis in the muscles. Although sodium barbital gradually inhibits the activity of CK with increased concentration, the inhibition effect can be completely reversed by dilution, indicating that the inactivation process is reversible. Detailed kinetics analysis, according to a previously presented theory, indicates that sodium barbital functions as a non complexing inhibitor, and its inhibition effect on CK is a slow reversible inactivation. In this study, a kinetic model of the substrate reaction is presented, and the microscopic rate constants for the reaction of sodium barbital with the free enzyme and the enzyme–substrate complexes are determined. Kinetic analysis reveals that sodium barbital might compete with both creatine and ATP, but mainly with creatine, to inhibit the activity of CK. The results suggest that CK might be a target for sodium barbital in vivo.Key words: creatine kinase; inactivation; kinetics; sodium barbital.

scholarly journals Human ‘creatine kinase conversion factor’ identified as a carboxypeptidase

1984 ◽  
Vol 221 (2) ◽  
pp. 465-470 ◽  
Author(s):  
R J Edwards ◽  
D C Watts
Keyword(s):  
Creatine Kinase ◽  
Conversion Factor ◽  
Electrophoretic Pattern ◽  
Beta Subunits ◽  
Rabbit Muscle ◽  
Activity Profile ◽  
Muscle Creatine Kinase ◽  
Electrophoretic Patterns ◽  
Carboxypeptidase N ◽  
Low Activity

The effect of partially purified ‘creatine kinase conversion factor’ on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.

scholarly journals Chaperone-Like Effect of the Linker on the Isolated C-Terminal Domain of Rabbit Muscle Creatine Kinase

2012 ◽  
Vol 103 (3) ◽  
pp. 558-566 ◽  
Author(s):  
Zhe Chen ◽  
Xiang-Jun Chen ◽  
Mengdie Xia ◽  
Hua-Wei He ◽  
Sha Wang ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Terminal Domain ◽  
Muscle Creatine
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