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.
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ABSTRACT:
SUBMITTER: Price NC
PROVIDER: S-EPMC1163623 | biostudies-other | 1982 Jan
REPOSITORIES: biostudies-other
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Project description:The refolding of rabbit muscle pyruvate kinase after denaturation by guanidine hydrochloride was studied. On dilution of the denaturing agent, enzyme activity is only partially regained. The extent of regain of activity is dependent on protein concentration, showing a marked decrease at higher concentrations. The failure to regain complete activity appears to be related to the formation of inactive aggregates, which can be separated from active enzyme by gel filtration. Insoluble aggregates can be partially re-activated after solubilization in guanidine hydrochloride. Changes in the circular-dichroism and fluorescence spectra during refolding suggest that a partially folded, inactive species is formed rapidly; this differs from native enzyme in being more susceptible to proteolysis by trypsin.
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Project description: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.
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Project description:Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle. This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP. The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme. Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions. Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes. This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA. However, 30 S particles failed to refold the enzyme.
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