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  • 1
    Electronic Resource
    Electronic Resource
    Reactivation and Refolding of Reassociated Dimers of Rabbit Muscle Creatine Kinase (2000)
    Springer
    The protein journal 19 (2000), S. 185-191 
    Details
    ISSN: 1573-4943
    Keywords: Creatine kinase ; dissociation ; reassociation ; hybrid dimer ; reactivation ; refolding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a good model for studying dissociation and reassociation during unfolding and refolding. This study compares self-reassociated CK dimers and CK dimers that contain hybrid dimers under proper conditions. Creatine kinase forms a monomer when denatured in 6 M urea for 1 h which will very quickly form a dimer when the denaturant is diluted under suitable conditions. After modification by DTNB, CK was denatured in 6 M urea to form a modified CK monomer. Dimerization of this modified subunit of CK occurred upon dilution into a suitable buffer containing DTT. Therefore, three different types of reassociated CK dimers including a hybrid dimer can be made from two different CK monomers in the proper conditions. The CK monomers are from a urea-denatured monomer of DTNB-modified CK and from an unmodified urea dissociated monomer. Equal enzyme concentration ratios of these two monomers were mixed in the presence of urea, then diluted into the proper buffer to form the three types of reassociated CK dimers including the hybrid dimer. Reassociated CK dimers including all three different types recover about 75% activity following a two-phase course (k 1 = 4.88 × 10−3 s−1, k 2 = 0.68 × 10−3 s−1). Intrinsic fluorescence spectra of the three different CK monomers which were dissociated in 6 M urea, dissociated in 6 M urea after DTNB modification, and a mixture of the first two dissociated enzymes were studied in the presence of the denaturant urea. The three monomers had different fluorescence intensities and emission maxima. The intrinsic fluorescence maximum intensity changes of the reassociated CK dimers were also studied. The refolding processes also follow biphasic kinetics (k 1 = 3.28 × 10−3 s−1, k 2 = 0.11 × 10−3 s −1) after dilution in the proper solutions. Tsou's method [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381–436] was also used to measure the kinetic reactivation rate constants for the different three types of reassociated CK dimers, with different kinetic reactivation rate constants observed for each type. CK dissociation and reassociation schemes are suggested based on the results.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007051619017
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  • 2
    Electronic Resource
    Electronic Resource
    Effect of Mg2+ During Reactivation and Refolding of Guanidine Hydrochloride-Denatured Creatine Kinase (2000)
    Springer
    The protein journal 19 (2000), S. 193-198 
    Details
    ISSN: 1573-4943
    Keywords: Creatine kinase ; magnesium ion ; reactivation ; refolding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296–1302; Yao et al. (1984), Biochemistry 23, 2740–2744; Yao et al. (1982), Sci. Sin. 25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg2+ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg2+ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg2+; however, the rates of reactivation are independent of the Mg2+ concentration. In addition, Mg2+ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg2+ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg2+ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007003703087
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  • 3
    Electronic Resource
    Electronic Resource
    Effects of Zinc on Creatine Kinase: Activity Changes, Conformational Changes, and Aggregation (2000)
    Springer
    The protein journal 19 (2000), S. 553-562 
    Details
    ISSN: 1573-4943
    Keywords: Creatine kinase ; zinc ; activation and inactivation ; conformational change ; aggregation ; Alzheimer's disease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007142117037
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    Paper (German National Licenses)
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