Kinetics of in vitro reconstitution of oligomeric enzymes by cross-linking

Hermann, Reinhard; Rudolph, Rainer; Jaenicke, Rainer

doi:10.1038/277243a0

Cited by 57 publications

(26 citation statements)

References 13 publications

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“…Apart from the approaches mentioned in the introduction [2,4,6,7], activity transport in the ultracentrifuge [21] and chemical modification [22,23] deserve mentioning here. In summarizing all available evidence, it may be concluded that in the case of lactic dehydrogenase the tetramer is the only active species; neither the monomer nor the dimer show catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

“…The bimolecular step involved in the reactivation of tetrameric enzymes (which clearly contradicts subunit activity [S]) may be attributed to the rate-limiting formation of either dimers or tetramers. Attempts to decide this alter-native using cross-linking of the reconstituting enzyme, have shown that, in the case of lactic dehydrogenase, reactivation and tetramer formation run parallel [6]. Independent evidence along this line may be gained from comparative reconstitution experiments on enzymes with identical catalytic function but different quaternary structure, e.g.…”

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confidence: 98%

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Dimeric Intermediates in the Dissociation of Lactic Dehydrogenase

1981

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Rate-determining second-order reactions in the reconstitution of a number of oligomeric enzymes have shown that catalytic activity requires association.In order to answer the question whether in the case of tetrameric enzymes, e.g. lactic dehydrogenase, the bimolecular step belongs to the dimerization of the monomer or the dimer, attempts have been made to isolate intermediates of dissociation, and to study their reconstitution to the native tetramer. Varying concentrations of urea or guanidine . HCI were applied in order to labilize the native quaternary structure; incubation time, temperature and enzyme concentration were changed as additional variables.Both isoenzymes, M4 from pig and bovine skeletal muscle, and H4 from pig heart show separable steps in their denaturation profiles on changing the concentration of urea or guanidine . analysis. Increased temperature yields wrong aggregates rather than low-molecular-weight intermediates, causing a drastic decrease of reactivation. Reconstitution of the metastable dimeric intermediates to reestablish the native tetramer is characterized by the same rate constants which have been reported previously for the overall reconstitution, starting from denatured monomers [R. Jaenicke (1979) FEBS Symp. 52, 187-1981. In the case of the isoenzyme M4 from pig skeletal muscle (after incubation in 1 M guanidine . HCI) reconstitution follows a simple bimolecular mechanism, while in the case of H4 from pig heart sigmoidal reactivation profiles indicate a more complicated mechanism involving rate-determining first-order processes.The fact there is no significant difference between the kinetics of reactivation during the dimer --$ tetramer transition on the one hand, and the monomer --f tetramer overall reconstitution on the other, strongly suggests that the rate-determining step in the consecutive association reaction underlying reactivation as well as tetramer formation must be the dimerization of inactive dimers.The autonomous and spontaneous acquisition of the three-dimensional structure of oligomeric proteins consists of consecutive folding and association reactions of the constituent polypeptide chains. Local 'packing' within these chains is considered to provide the highly specific intersubunit contact areas of the monomers which then assemble to the native, biologically active oligomers [l]. Secondorder reactivation kinetics prove for a number of oligomeric enzymes that full catalytic activity necessitates association, although there is experimental evidence contradicting this finding [2 -51.The question of whether, in the case of oligomers with more than two subunits, like lactic dehydrogenase and a great number of other tetrameric enzymes, enzymatic activity requires complete assembly to the native quaternary structure, or whether intermediates of association are catalytically active is still open, even in cases where subunit activity can be excluded. The bimolecular step involved in the reactivation of tetrameric enzymes (which clearly contradicts subunit activity [S]) ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 98%

Dimeric Intermediates in the Dissociation of Lactic Dehydrogenase

1981

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“…0341-0382/81/0900-0772 $01.00/0 therefore proposed as a tool to study association reactions of oligomeric proteins [7]. Applying this m ethod we were able to show that the dim er -+ tetram er transition is rate-lim iting for the reconsti tution of lactic dehydrogenase from skeletal muscle; at the same tim e evidence was presented for a dissociation-association equilibrium between dimers and monomers [8].…”

Section: Introductionmentioning

confidence: 75%

“…The combined use of fast cross-linking with glutaraldehyde and subsequent SDS polyacrylamide gel-electrophoresis allows the quantitative analysis o f the reassociation o f oligomeric enzymes [7], Reconstitution of porcine skeletal muscle lactic de hydrogenase which was previously investigated by a TIME (s) Fig. 4.…”

Section: K Inetics O F Reassociationmentioning

confidence: 99%

Reassociation of Lactic Dehydrogenase from Pig Heart Studied by Cross-Linking with Glutaraldehyde

Bernhardt

Rudolph

Jaenicke

1981

Zeitschrift Für Naturforschung C

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Cross-linking with glutaraldehyde has been successfully applied in order to analyze the kinetics of reassociation of oligomeric enzymes (R. Hermann, R. Rudolph, and R. Jaenicke Nature 277, 243-245 (1979)). In the present study the assembly of lactic dehydrogenase from pig heart is investigated using this approach.In order to eliminate perturbations caused by excessive folding reactions, acid dissociation was performed in the presence of 0.8 M Na2S 0 4 at 0°C . Under optimum conditions complete crosslinking of the tetrameric enzyme was achieved in less than 2 minutes.Cross-linking during reconstitution proves the dimer to be the only intermediate of reasso ciation. The dimer -*■ tetramer transition is found to be rate-limiting for both reassociation and reactivation, suggesting the tetramer to be the enzymatically active species.The presence of monomers during reconstitution indicates that tetramer formation is preceded by a fast monomer-dimer equilibrium.The kinetic model describing the experimental data 4M^2D^Tis characterized by an equilibrium constant K = 3 ± 1 • 107 liter • m ol-1, and a second-order rate constant k = 1.4 ± 0.2 • 104 liter • mol-1 • s-1.

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“…Since the reconstitution of a tetrameric enzyme like mammalian lactic dehydrogenase is comprised of at least two association reactions, namely the dimerization of monomers to dimers and of dimers to tetramers [8], it could not be decided which of these reactions is the actual ratelimiting step in the reconstitution of the enzyme. A thorough comparison of reactivation and reassociation (determined by cross-linking of association intermediates with glutaraldehyde) reveals that a firstorder transconformation reaction participates as a rate-determining step in the kinetics of reactivation [2]. Reactivation experiments after dissociation at high hydrostatic pressure (B. C. Schade, R. Rudolph, H.-D. Liidemann and R. Jaenicke, unpublished results) have also shown that a first-order reaction contributes significantly to the overall process of reactivation.…”

Section: Resultsmentioning

confidence: 99%

Effects of Low Concentrations of Guanidine . HCl on the Reconstitution of Lactic Dehydrogenase from Pig Muscle in vitro. Evidence for Guanidine Binding to the Native Enzyme

1979

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The presence of low concentrations of guanidine . HC1 has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscle after preceding dissociation and deactivation in various denaturants. The observed attenuation is a function of the amount of guanidine . HC1 present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCI on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.Reactivation and reassociation of lactic dehydrogenase from pig muscle have been the subject of extensive studies, and the correlation of both processes is at present fairly well understood [l, 21. Therefore, this oligomeric enzyme is a suitable system for studying the influence of effectors on the process of folding and reconstitution. In a previous study the effect of NAD was investigated in order to elucidate the 'nucleating power' of coenzymes and substrates on the folding of the nascent polypeptide chain destabilizing agent. Guanidine has been frequently used as a strong denaturant for proteins [4,5], which have been shown to undergo pronounced structural disintegration at concentrations CG 2 1 M.Although the cooperativity and extent of denaturation have been extensively studied, only a little information is available about the binding of guanidine . HCl to enzymes at predenaturing concentrations, and its influence on intermediates of reconstitution. Effects of small amounts of guanidine . HC1 present when reactivation is initiated by diluting highly concentrated solutions of the denaturant are in general ignored. In the present study evidence is given for an influence of low concentrations of guanidine . HCI on the rate of reconstitution of lactic dehydrogenase from pig muscle. The effect is attributed to a significant binding of the denaturant to intermediates of reconstitution, as well as the native enzyme. Preliminary experiments using a number of other dehydrogenases prove that the effect cannot be generalized, either in qualitative or in quantitative terms.

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Kinetics of in vitro reconstitution of oligomeric enzymes by cross-linking

Cited by 57 publications

References 13 publications

Dimeric Intermediates in the Dissociation of Lactic Dehydrogenase

Dimeric Intermediates in the Dissociation of Lactic Dehydrogenase

Reassociation of Lactic Dehydrogenase from Pig Heart Studied by Cross-Linking with Glutaraldehyde

Effects of Low Concentrations of Guanidine . HCl on the Reconstitution of Lactic Dehydrogenase from Pig Muscle in vitro. Evidence for Guanidine Binding to the Native Enzyme

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