D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation of the Octameric Enolase from S. Pyogenes - One Interface Stabilizes Another

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO₄, a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO₄ dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable.     

Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase

The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated. Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions. The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein. Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics. In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state. Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers. The reactivation yield under the in vitro conditions used follows an optimum behavior.     

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca²⁺-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.     

Purification, characterization, and distribution of enolase isozymes in chicken

Chicken brain enolase was found to show multiple forms (I, II and III) separable by DEAE-cellulose column chromatography, whereas enolase from chicken skeletal muscle showed a single form. Brain enolase I, enolase III and muscle enolase were purified to electrophoretic homogeneity. These three isozymes were dimeric enzymes, each being composed of two identical subunits, alpha, gamma and beta, having molecular weight of 51,000 +/- 600, 52,000 +/- 550 and 51,500 +/- 650, respectively, as determined by SDS-polyacrylamide gel electrophoresis analysis. Brain enolases I, II and III and muscle enolase had similar catalytic parameters, including almost the same Km values and pH optima. Specific antibodies against brain enolase I, enolase III and muscle enolase, raised in rabbit, showed no cross-reactivity with each other. Antibodies for brain enolases I and III also reacted with brain enolase II, indicating that brain enolase II was the hybrid form (alpha gamma) of brain enolases I (alpha alpha) and III (gamma gamma). Enolases from chicken liver, kidney and heart reacted with the antisera for brain enolase I, but not with those for brain enolase III or muscle enolase. Developmental changes in enolase isozyme distribution were observed in chicken brain and skeletal muscle. In brain, the alpha gamma and gamma gamma forms were not detected in the early embryonic stage and increased gradually during the development of the brain, whereas the alpha alpha form existed at an almost constant level during development. In skeletal muscle, complete switching from alpha alpha enolase to beta beta was observed during the period around hatching.     

Dimeric form of diphtheria toxin: purification and characterization

Many preparations of diphtheria toxin were found to contain dimeric and multimeric toxin forms. The monomeric and dimeric forms were fractionated to greater than 98% purity, and their properties were compared. Dimeric toxin slowly dissociated to native monomers in solution at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. In cell culture assays and rabbit skin tests, the dimer exhibited no significant toxic activity, except for that attributable to trace contamination by monomer, or partial dissociation to monomer during the incubation period. In guinea pig lethality tests, however, toxic activity varied depending upon the dose. At least 7-fold greater amounts of dimer than monomer (161 ng vs. 22 ng, respectively) were required to cause death at 18 h, whereas similar weights of the two toxin forms (22 ng) caused death at 120 h. This variability probably reflected slow dissociation of dimer to monomer in the animal. The dimer was unable to bind toxin receptors on the surface of susceptible cells, whereas it retained full activity in the ADP-ribosyltransferase, NAD-glycohydrolase, or ligand-binding assays. Thus, the lack of toxicity of the dimeric toxin may have resulted from distortion or occlusion of the receptor binding site on the B moiety. We propose that the dimer contains two monomeric units bound by hydrophobic interactions and that the points of contact involve regions of the B moieties that are normally buried in the native monomer.     

Effects of the G376E and G157D mutations on the stability of yeast enolase--a model for human muscle enolase deficiency

The first known human enolase deficiency was reported in 2001 [Comi GP, Fortunato F, Lucchiari S, Bordoni A, Prelle A, Jann S, Keller A, Ciscato P, Galbiati S, Chiveri L et al. (2001) Ann Neurol50, 202-207]. The subject had inherited two mutated genes for beta-enolase. These mutations changed glycine 156 to aspartate and glycine 374 to glutamate. In order to study the effects of these changes on the structure and stability of enolase, we have introduced the corresponding changes (G157D and G376E) into yeast enolase. The two variants are correctly folded. They are less stable than wild-type enolase with respect to thermal denaturation, and both have increased Kd values for subunit dissociation. At 37 degrees C, in the presence of salt, both are partially dissociated and are extensively cleaved by trypsin. Under the same conditions, wild-type enolase is fully dimeric and is only slightly cleaved by trypsin. However, wild-type enolase is also extensively cleaved if it is partially dissociated. The identification of the cleavage sites and spectral studies of enolase have revealed some of the structural differences between the dimeric and monomeric forms of this enzyme.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric Cα structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

The pressure-induced inactivation of mammalian enolases is accompanied by dissociation of the dimeric enzyme

The effects of exposure to pressure on both the activity and the quaternary structure of rabbit brain enolases, forms alpha alpha, alpha gamma, and gamma gamma were studied in the pressure range of 1 to 3400 bar. Effects on quaternary structure were determined by subunit scrambling (the formation of alpha alpha and gamma gamma from alpha gamma or vice versa). All three dimers are stable up to pressures of 1200 bar. The dissociation of gamma gamma begins at 1200 bar, yielding a stable monomer; inactivation of gamma gamma does not begin until the pressure is greater than 2000 bar. Dissociation of gamma gamma is not accompanied by changes in the tryptophan fluorescence of the protein. However, the fluorescence does decrease when the pressure is greater than 2000 bar, the point at which inactivation of gamma gamma starts. The alpha monomer, on the other hand, is unstable in the pressure range that produces dissociation of alpha alpha. This process, which also begins at 1200 bar, is paralleled by inactivation. Crosslinking the enzyme with glutaraldehyde demonstrated that the inactive form of the enzyme is monomeric. The pressure-induced inactivation of these forms of enolase is thus clearly a two-step process, with both dissociation and inactivation occurring. The difference in pressure sensitivity of rabbit brain alpha alpha and gamma gamma is due to a difference in stability of the alpha and gamma monomers and not due to a difference in the pressures required for dissociation.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric C structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases

Two isozymes of enolase, alpha alpha and gamma gamma, have been purified from rabbit brain and characterized. The kinetic properties of alpha alpha and gamma gamma (pH optimum, Km for phosphoglycerate and phosphoenolpyruvate, requirement for a divalent cation) are very similar to those of rabbit enolase, form beta beta, and to those of enolase isozymes from other species. However, several novel properties were observed. (i) All the enolases studied were inhibited by Na+ and Li+. (ii) The rabbit enolases, but not yeast enolase, were activated by K+, NH4+, Cs+, and Rb+. (iii) Rabbit enolase is more susceptible to inhibition by excess Mg2+ than is the yeast enolase; the increased inhibition by Mg2+ above pH 7.1 accounts, at least in part, for the observed differences between mammalian and yeast enolases in their pH optima for activity.     

Structural and Mechanical Hierarchies in the α-Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.     

Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system

The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation.     

The Concave Face of Decorin Mediates Reversible Dimerization and Collagen Binding

Decorin, the prototypical small leucine-rich proteoglycan, binds to collagen and thereby regulates collagen assembly into fibrils. The crystal structure of the decorin core protein revealed a tight dimer formed by the association of two monomers via their concave faces (Scott, P. G., McEwan, P. A., Dodd, C. M., Bergmann, E. M., Bishop, P. N., and Bella, J. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 15633–15638). Whether decorin binds collagen as a dimer has been controversial. Using analytical ultracentrifugation, we determined a dissociation constant of 1.37 ± 0.30 μm for the mouse decorin dimer. Dimerization could be abolished by engineering glycosylation sites into the dimer interface; other interface mutants remained dimeric. The monomeric mutants were as stable as wild-type decorin in thermal unfolding experiments. Mutations on the concave face of decorin abolished collagen binding regardless of whether the mutant proteins retained the ability to dimerize or not. We conclude that the concave face of decorin mediates collagen binding and that the dimer therefore must dissociate to bind collagen.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.     

The pathway by which the tetrameric protein transthyretin dissociates

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct dimer interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into dimers along either of the two unique quaternary interfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)(2) construct, affording (TTR-S-S-TTR)(1) retaining a stable 16-stranded beta-sheet structure that is equivalent to the dimer not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)(2) affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)(2) construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)(2) construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C(2) axis affording AB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/CD dimer-dimer interface impose kinetic stabilization upon TTR and disease-associated variants thereof.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Evidence for the activity of immobilised monomers of triose phosphate isomerase.

Chicken muscle triose phosphate isomerase was immobilised by attachment to Sepharose 4B. The immobilised dimeric enzyme was dissociated with guanidinium chloride to yield bound monomeric triose phosphate isomerase. This regained activity on removal of the denaturant, showing that isolated monomers possess activity; the apparent K_m of the immobilished subunits was the same as that of the immobilised dimers. Under appropriate conditions, it was possible to rehybridise the immobilised monomers to native dimers, and also to form a hybrid dimer from the chicken muscle and rabbit muscle enzymes.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.