The effect of limited proteolysis on rabbit muscle creatine kinase

Price, Nicholas C.; Murray, Stephen A.; Milner‐White, E. James

doi:10.1042/bj1990239

Cited by 22 publications

(16 citation statements)

References 23 publications

(27 reference statements)

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“…Experiments involving cross-linking of the proteolysed enzyme have shown that, under non-denaturing conditions, the small and large fragments remain associated (Price et al, 1981). When the fragments are separated by treatment with denaturing agents such as guanidine hydrochloride, the ability to refold on dilution of the denaturing agent is greatly impaired.…”

Section: Discussionmentioning

confidence: 99%

The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding

Price¹,

Stevens²

1982

Biochemical Journal

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A number of aspects of the refolding of denatured rabbit muscle creatine kinase have been studied. Addition of substrates has no effect on the rate or extent of regain of activity. The changes in protein fluorescence during refolding broadly parallel the regain of activity. A study of the susceptibility of the enzyme to proteolysis during refolding indicates that there is no significant accumulation of folded, but inactive, intermediates in the folding process. Modification of the reactive thiol group on each subunit of the enzyme by small reagents such as iodoacetate or iodoacetamide prior to denaturation has only a small effect on the rate of subsequent refolding. However, modification by the bulky reagent 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate has a very large effect on the ability of the enzyme to refold after denaturation.In a previous paper it has been shown that the dimeric enzyme creatine kinase (EC 2.7.3.2) from rabbit muscle which had been denatured in 3M-guanidine hydrochloride could refold on dilution of the denaturing agent (Bickerstaff et al., 1980). The refolded enzyme appeared to be identical with the native enzyme in terms of catalytic activity, kinetic parameters, c.d. spectrum and electrophoretic behaviour. From studies of the rate of regain of activity, of the decline in the number of reactive (exposed) thiol groups and of the ability of subunits to be cross-linked, a minimal mechanism for the refolding process was proposed. This mechanism consisted of a rapid phase (complete within about 15min) involving refolding and reassociation of subunits with a regain of approx. 70% of activity. The remaining activity was regained in a slower process lasting several hours.In the present paper, we report the results of experiments in which we have attempted to detect intermediates in the early part of the refolding process. The results obtained suggest that the Abbreviations used: SDS, sodium dodecyl sulphate;

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Section: Discussionmentioning

confidence: 99%

The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding

Price¹,

Stevens²

1982

Biochemical Journal

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“…This is consistent with an equilibrium unfolding study of proteinse K‐nicked MM‐CK. Although native MM‐CK is digested with proteinase K at a unique site in a large surface loop between strands β9 and β10, the fragments remain associated below 0.9 M GdmCl 4, 36–38…”

Section: Resultsmentioning

confidence: 70%

Microwave Driven Bray–Liebhafsky Oscillatory Reaction

2004

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Complex systems, far from thermodynamic equilibrium, can show unusual dynamic behavior. [1,2] The reaction between hydrogen peroxide and potassium iodate in acidic water solution, the Bray ± Liebhafsky (BL) reaction, [3,4] is one such system. Taking the energy of decomposed peroxide as the driving force, the reaction evolves in time through oscillatory dynamics. Reaction intermediates periodically form and decay by the periodic channeling of energy into the two reaction pathways (branches) [3,4] shown in Equations (1) and (2).The overall effect is the decomposition of hydrogen peroxide, as shown in Equation (3)Formal kinetic investigations of the processes shown in Equations (1) and (2) [5] during the course of the reaction have shown a higher activation energy for Equation (2). This is in agreement with the observation [4,6,7] that the direct oxidation of iodine (without any iodate present initially) is a rather slow process. As a result, iodine initially accumulates during the prevalence of the process of iodate reduction, Equation (1). Despite the higher activation energy, the process of iodine oxidation, Equation (2), periodically dominates the reduction branch with a speed that exceeds the speed of the reduction branch by 20 ± 30-times. [8] This causes oscillations in the concentration of iodine and other intermediates involved in the complex processes shown in Equations (1) and (2).Reactions comprising the reduction branch, Equation (1), in which hydrogen peroxide is being oxidized are relatively well rationalized.[9±13] A detailed model explaining why the energy is periodically channeled in Equation (2)-in actually creating the necessary reactive intermediates-despite its higher activation energy is missing. Understanding the switching mechanism between the BL reaction branches is of great importance. It would allow a more efficient control of processes in living organisms, where oscillatory evolutions (on a very broad time scale) are inherent phenomena. [14] [6] a) H. E. Katz, A. J. Lovinger, J. Johnson, C. Kloc, T.

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“…We also need to explain why the two peptides remain firmly associated after proteinase K treatment, requiring urea or SDS treatment to dissociate them (Price et al, 1981). Since there are no disulphide bridges in CK (Watts, 1973), such a strong interaction is likely to be hydrophobic.…”

Section: Monoclonal-antibody Studies Of Creatine Kinasementioning

confidence: 99%

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site

Morris¹,

Frost²,

Head³

1985

Biochemical Journal

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Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.

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The effect of limited proteolysis on rabbit muscle creatine kinase

Cited by 22 publications

References 23 publications

The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding

The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding

Microwave Driven Bray–Liebhafsky Oscillatory Reaction

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site

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