Isoenzyme-specific thermostability of human cytosolic creatine kinase

Gao, Yan-Song; Zhao, Tong‐Jin; Chen, Zhe; Li, Chang; Wang, Yin; Yan, Yong‐Bin; Zhou, Hai‐Meng

doi:10.1016/j.ijbiomac.2010.03.025

Cited by 17 publications

(37 citation statements)

References 32 publications

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“…Our results suggest that recombinant CKBB began to be partially inactivated after 10 min heat-treatment at 38 °C, a temperature close to the normal body temperature. As reported by [2] On the other hand, thermally inactivated CKBB has been shown to have better results on activity recovery than CKMM. Up to >60% of activity could be regained after reactivation on ice for 12 h for the enzymes treated at temperatures above 50 °C [2].…”

Section: Resultsmentioning

confidence: 56%

“…Oxidative modifications have a major role in the pathogenesis of neurodegenerative disorders, regulated by protein and lipid oxidative modifications [20]. Studies on human muscle creatine kinase have confirmed that thermal irreversibility of the enzyme was reached under the temperature of 56 °C [2]. Western blotting with the antibodies against CK isoenzyme using cell homogenate from HeLa cells identifies a single band of 43 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…CK possesses four cysteine residues and only one of them is believed to be essential for catalytic activity, where the other three are thought to be involved in CK folding [25][26][27]. According to [2] muscle isoform of CK could retain its activity at temperatures below 50 °C and being completely in-activated after heat treatment at temperatures above 62 °C [28,29]. Although, CKBB has a high percentage of the primary sequence similarity with CKMM, the thermal stability of human CKBB is substantially lower than that of CKMM, and the midpoint temperatures of thermal inactivation decreased about 15 °C [2].…”

Section: Resultsmentioning

confidence: 99%

“…According to [2] muscle isoform of CK could retain its activity at temperatures below 50 °C and being completely in-activated after heat treatment at temperatures above 62 °C [28,29]. Although, CKBB has a high percentage of the primary sequence similarity with CKMM, the thermal stability of human CKBB is substantially lower than that of CKMM, and the midpoint temperatures of thermal inactivation decreased about 15 °C [2]. Our results suggest that recombinant CKBB began to be partially inactivated after 10 min heat-treatment at 38 °C, a temperature close to the normal body temperature.…”

Section: Resultsmentioning

confidence: 99%

“…CKBB is a homodimer enzyme, catalyzing reversible transfer of γ-phosphate group from ATP molecule to creatine, with formation of buffering phosphocreatine (PCr) and ADP. Domains associated with ATP production and consumption are predominantly connected by phosphocreatine/ creatine network [2]. CK isoforms accumulate PCr within the cell to high concentrations and uses this molecule in the reverse reaction to regenerate pools of ATP under increased workload [3].…”

Section: Also Examination Of Immunoblotted Membrane Patterns By Sdmentioning

confidence: 99%

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Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide

Rakhmetov¹

2015

Ukr.Biochem.J.

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Section: Resultsmentioning

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Also Examination Of Immunoblotted Membrane Patterns By Sdmentioning

confidence: 99%

See 3 more Smart Citations

Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide

Rakhmetov¹

2015

Ukr.Biochem.J.

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Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

et al. 2013

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The Fc-III tag is a newly developed fusion tag that can be applied to protein purification and detection. In the present work, we use the Fc-III-tagged green fluorescent protein (GFP) and human muscle creatine kinase (CK) as model systems to investigate effects of the Fc-III tag on activities and stabilities of the expressed multicysteine-containing proteins. Our results show the Fc-III tag has no adverse effects on the fluorescence of GFP and reduces the occurrence of GFP misfolding due to incorrect Cys oxidation compared with the His-tagged protein. The activity and stability of the Fc-III-tagged CK is slightly lower than that of the tag-free CK, but is higher than that of the His-tagged CK as determined by the ratio of the oxidized versus reduced CK. A major portion of His-tagged CK is in its oxidized form, while that of the Fc-III-tagged CK is in its reduced form. A folding model of CK with different tags was proposed, which may provide insights into the effect of the Fc-III tag on the conformations of disulfide-bridged proteins.

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Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System

Fan

Liu

Chang

et al. 2012

Appl Biochem Biotechnol

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In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k (2) value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8 × 10(-5) s(-1). The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.

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Isoenzyme-specific thermostability of human cytosolic creatine kinase

Cited by 17 publications

References 32 publications

Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide

Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide

Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System

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