kinetics of reactivation of rabbit muscle aldolase after denaturation and dissociation in various solvent media

The denaturation of aldolase from rabbit muscle in various solvents leads to significant qualitative and quantitative differences with respect to the structural disintegration of the enzyme. The differences refer to the quaternary structure and to the conformation which is changed only slightly in MgCl₂ while in guanidine · HCl or urea at pH 2 the molecule is close to the state of the random coil.Using the enzymic activity as a quantitative measure for the refolding process, the reaction order and the rate constants of the processes of structure formation ( _i N^*) are found to be identical.This observation suggests a common intermediate D in the process of renaturation after denaturation and dissociation in the different solvent media. D may be considered an intermediate state with a defined number of nucleation centers whose rapid formation is predetermined by the aminoacid sequence.As taken from the first order kinetics in the given range of enzyme concentration, transconformation reactions are rate limiting in the obligatory pathway of refolding. At low enzyme concentrations second order steps gain importance which indicates that the enzymic activity is significantly modified by the formation of the native quaternary structure.Dedicated to Professor J. M. Sturtevant     

Equilibrium partition studies of the interaction between aldolase and myofibrils

The adsorption of aldolase to myofibrils derived from rabbit skeletal muscle has been investigated by partition equilibrium studies at pH 6.8, I = 0.158 M, and the results interpreted in terms of an intrinsic association constant of 410,000 m^?1 for the interaction of four sites on aldolase with myofibrillar sites, there being one such site for every 10–12 heptameric repeat units of F-actin-tropomyosin-troponin thin filament. Involvement of the active site of the enzyme in the adsorption process is indicated by the fact that competitive inhibition of the phenomenon by phosphate may be accounted for by an intrinsic association constant of 400 m^?1 for the aldolase-phosphate interaction, a value in good agreement with that describing phosphate inhibition of the enzymatic hydrolysis of fructose-1,6-bisphosphate under similar conditions. On the basis of these equilibrium constants plus the aldolase and thin filament contents of muscle, resting muscle is indicated as containing a significant proportion (25–30%) of aldolase in the bound form, with changes in the subcellular distribution of the enzyme being likely during exercise due to the increased concentrations of Ca²⁺ and fructose-1,6-bisphosphate that then prevail.     

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.     

Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.     

Structural studies on acid unfolding and refolding of recombinant human interferon gamma

Interferon gamma is distinguished from other types of interferons in its instability upon acid treatment, as demonstrated by a loss of antiviral activity. Acid unfolding and refolding experiments were performed with recombinant DNA derived human interferon gamma. When the protein was subjected to unfolding and refolding, the refolded protein showed two peaks (peaks I and II) in gel filtration which have been shown to differ in size, structure, and antiviral activity. When the smaller, peak II, form was unfolded by dialysis against 0.01 M HCl containing 0.1 M NaCl (pH 2) and refolded by dialysis against various solvents at neutral pH, it re-formed as peak II but also generated peak I, and the ratio of the two forms was dependent on protein concentration and solvent conditions. Higher protein concentrations and higher ionic strength led to a greater ratio of peak I to peak II. Phosphate buffers caused precipitation of peak I. Since peak II is 4-8 times more active than peak I in the antiviral bioassay, generation of peak I by acid treatment of peak II should lead to a decrease in antiviral activity.     

Identification of the C-1-phosphate-binding arginine residue of rabbit-muscle aldolase. Isolation of 1,2-cyclohexanedione-labeled peptide by chemisorption chromatography.

The arginine-specific reagent 1,2-cyclohexanedione reacts selectively with the arginine residue of the C-1-phosphate-binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu-43 to Arg-56, the residue modified by cyclohexanedione being Arg-55. This peptide was absent form digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg-55 with loss of activity. Selective isolation ofthe peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix-bound boric acid groups with vicinal cis-hxdroxyl groups of cyclohexanedione-modified arginine side chains.     

Refolding and reactivation of liver alcohol dehydrogenase after dissociation and denaturation in 6M guanidine hydrochloride.

Horse-liver alcohol dehydrogenase has been dissociated and denatured by 6 M guanidinium hydrochloride. Removal of the denaturant under optimum conditions of the solvent leads to partial reactivation. The concentrations of the enzyme, as well as the coenzyme (NAD+), and Zn2+, affect the reactivation significantly, since high concentrations promote the formation of inactive aggregation products. Analyzing the kinetics of reactivation and reassociation, conditions far from equilibrium of dissociation-association provide maximum yields (approximately 70%). The sigmoidal kinetic traces suggest a superposition of first-order transconformation and second-order association reactions; the latter are corroborated by the concentration dependence of the reactivation reaction. The coenzyme, NAD+, has no influence on the kinetics of reactivation. Addition of Zn2+ leads to a significant decrease of the rate and yield of reactivation. The process of renaturation, as reflected by the regain of native fluorescence shows complex kinetics: rapid relaxations are followed by slower first-order and second-order processes which parallel reactivation.     

Subunit interactions of rabbit muscle aldolase. Conformational change of aldolase in magnesium chloride and its relationship to the dissociation of subunits.

The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA³. The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg²⁺ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.     

Equilibrium dissociation and unfolding of the Arc repressor dimer

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.