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

Abstract

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;

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfishmacrobrachium vollenhovenii (herklots)

“…Aggregation of unfolded enzymes had been reported in many cases of denaturation/renaturation experiments [14,43]. The remarkable thing about the renaturation of the denatured crayfish GST was that the refolding of the completely unfolded structures to monomeric species, which subsequently associated with catalytically active dimers, was a very rapid process as had been noted in many other cases of renaturation experiments [41,43,44].…”

Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding

“…CK, however, is a large enzyme likely to consist of several domains and renaturation of activity occurs over several minutes ( Fig. 7; Price & Stevens, 1982;Zhou & Tsou, 1986). It would seem certain that some refolding, at least of secondary structure, occurs before activity recovers as a result of a 'final' conformational change (Watts, 1973;Price & Stevens, 1982;Zhou & Tsou, 1986).…”

“…The enzyme undergoes a significant and essential conformational change on binding its substrates (Watts, 1973), a change which can be detected by its effects on antibody binding (Samuels, 1961). Under appropriate conditions, CK will refold after denaturation by urea or guanidine and will recover enzymic activity (Price & Stevens, 1982).…”

Dissecting the key residues crucial for the species-specific thermostability of muscle-type creatine kinase

“…Muscle-type CK (MMCK) participates in muscle contraction by specifically binding with the M-line during activation [17][18][19]. Due to its important physiological functions, CK has been studied extensively, particularly its catalytic mechanism [13,20], structure [20], regulation [17,21,22], thermostability [23][24][25][26][27], folding [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] and its roles in diseases [21,37,[43][44][45][46].…”