Reassociation Rate Research Articles

Phosphorylation of NONPHOTOTROPIC HYPOCOTYL3 affects photosensory adaptation during the phototropic response.

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light (BL) photoreceptor phototropin 1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here, we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We generated unphosphorable and phosphomimetic NPH3 proteins on seven phosphorylation sites in the etiolated seedlings of Arabidopsis. Unphosphorable NPH3 showed a shortening of its retention time in the cytosol and caused an inability to adapt to very low fluence rates of BL (∼10-5 µmol m-2 s-1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened retention time in the cytosol and could not enable the adaptation to BL at fluence rates of 10-3 µmol m-2 s-1 or more. Our results indicate that the activation level of phot1 and the corresponding phosphorylation level of NPH3 determine the dissociation rate and the reassociation rate of NPH3 on the plasma membrane, respectively. These mechanisms may moderately maintain the active state of phot1 signaling across a broad range of BL intensities and contribute to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls.

Energy-Efficient Sleep Strategy With Variant Sleep Depths for Open-Access Femtocell Networks

This letter aims to enhance the energy-efficiency (EE) for hyper-dense small-cell networks by designing a low-complexity sleep strategy for base-stations (BSs). First, four operation modes are considered for BSs, i.e., active, standby, sleep, and off. To be specific, the active, standby, and sleep modes are for serving the delay-intolerant, delay-sensitive, and rate-sensitive demands. Second, the BSs with lower EE are iteratively considered to switch into lower-energy operation modes, if all the users with stringent delay requirement can be reassociated with the quality-of-service (QoS) provision. To improve the success rate of reassociation, all the BSs that can reach the predefined QoS can be the feasible targets. Compared with the conventional schemes, a remarkable 72% and 51% enhancement of EE can be achieved and more BSs can operate in the lower-energy modes.

Synthesis and characterization of citric acid esterified rice starch by reactive extrusion: A new method of producing resistant starch

Citric acid-esterified rice starch was synthesized using a one-step reactive extrusion method. The structure, physicochemical properties, and digestibility of the esterified starch were then measured. An in vitro digestion study indicated that esterification significantly increased the level of resistant starch (RS) in the samples. Fourier transform infrared spectroscopy (FT-IR) confirmed esterification had occurred between the citric acid and starch, with a new peak appearing at around 1730 cm−1. X-ray diffraction indicated that esterification caused a decrease in the degree of crystallinity in the starch. The swelling power and solubility of starch citrate were both lower than unmodified starch. Turbidity measurements suggested there was a slower rate of reassociation in starch citrates during storage. These results suggest that citric acid esterification promoted cross-linking within the starch that significantly reduced its digestibility. This simple method may be useful for the creation of healthier starchy foods.

RNA-DNA fibers and polygons with controlled immunorecognition activate RNAi, FRET and transcriptional regulation of NF-κB in human cells.

Nucleic acid–based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible ‘smart’ devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA–DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.

Disentangling Ligand Effects on Metathesis Catalyst Activity: Experimental and Computational Studies of Ruthenium-Aminophosphine Complexes.

Second-generation ruthenium olefin metathesis catalysts bearing aminophosphine ligands were investigated with systematic variation of the ligand structure. The rates of phosphine dissociation ( k1; initiation rate) and relative phosphine reassociation ( k-1) were determined for two series of catalysts bearing cyclohexyl(morpholino)phosphine and cyclohexyl(piperidino)phosphine ligands. In both cases, incorporating P-N bonds into the architecture of the dissociating phosphine accelerates catalyst initiation relative to the parent [Ru]-PCy3 complex; however, this effect is muted for the tris(amino)phosphine-ligated complexes, which exhibit higher ligand binding constants in comparison to those with phosphines containing one or two cyclohexyl substituents. These results, along with X-ray crystallographic data and DFT calculations, were used to understand the influence of ligand structure on catalyst activity. Especially noteworthy is the application of phosphines containing incongruent substituents (PR1R'2); detailed analyses of factors affecting ligand dissociation, including steric effects, inductive effects, and ligand conformation, are presented. Computational studies of the reaction coordinate for ligand dissociation reveal that ligand conformational changes contribute to the rapid dissociation for the fastest-initiating catalyst of these series, which bears a cyclohexyl-bis(morpholino)phosphine ligand. Furthermore, the effect of amine incorporation was examined in the context of ring-opening metathesis polymerization, and reaction rates were found to correlate well with catalyst initiation rates. The combined experimental and computational studies presented in this report reveal important considerations for designing efficient ruthenium olefin metathesis catalysts.

Identification of drivers for the metamorphic transition of HIV-1 reverse transcriptase.

Recent structural characterizations of the p51 and p66 monomers have established an important starting point for understanding the maturation pathway of the human immunodeficiency virus (HIV)-1 reverse transcriptase p66/p51 heterodimer. This process requires a metamorphic transition of the polymerase domain leading to formation of a p66/p66' homodimer that exists as a structural heterodimer. To better understand the drivers for this metamorphic transition, we have performed NMR studies of 15N-labeled RT216 - a construct that includes the fingers and most of the palm domains. These studies are consistent with the conclusion that the p66 monomer exists as a spring-loaded complex. Initial dissociation of the fingers/palm : connection complex allows the fingers/palm to adopt an alternate, more stable structure, reducing the rate of reassociation and facilitating subsequent maturation steps. One of the drivers for an initial extension of the fingers/palm domains is identified as a straightening of helix E relative to its conformation in the monomer by eliminating a bend of ∼50° near residue Phe160. NMR and circular dichroism data also are consistent with the conclusion that a hydrophobic surface of palm domain that becomes exposed after the initial dissociation, as well as the intrinsic conformational preferences of the palm domain C-terminal segment, facilitates the formation of the β-sheet structure that is unique to the active polymerase subunit. Spectral comparisons based on 15N-labeled constructs are all consistent with previous structural conclusions based on studies of 13C-methyl-labeled constructs.

Quantitative Study of As (V) and As (III) Interaction with Mangrove DNA by Molecular Fluorescence Spectroscopy

This study describes the in vitro study of (1:1) one step nucleophilic displacement ([Formula: see text]) of phosphate by heavier anion arsenate and arsenite in the DNA of arsenic ridden Sundarban mangroves. Mangrove DNA was found to give rise to a broad fluorescence and its integrated fluorescence intensity was enhanced on addition of As (V) and As (III), respectively. Analyses of the fluorescence parameter showed adequacy of 1:1 model to describe substitution of phosphate of mangrove DNA chain exiplex by arsenate and arsenite with equilibrium constant (log Kc) ranging between 4.19 and 4.32 for As (V), and between 3.77 and 3.89 for As (III) at pH 7 and 25°C. In the cases, the melting temperature (Tm) and reassociation rate constant of mangrove DNA was increased on treatment with As (V) and As (III). It is suggested that heavier ion arsenate and arsenite may substitute phosphate in natural DNA.

Mathematical model of colonization process of mycorrhizal plants: effect of interaction between plants with fungi

In interspecific mutualisms, the interacting species generally disperse independently, by which the colonization must be restricted by the reassociation between them. We theoretically analyzed colonization process of mycorrhizal plant in a novel habitat, focusing on the probability of colony establishment under environmental fluctuation. The process is a joint process of two sub-processes before and after the association between the plant and the mycorrhizal fungi. Properties of whole system are influenced by five parameters, means and variances of colony growth rates of two sub-systems, and a reassociation rate. Our analysis revealed that (1) the mean colony growth rates of the host alone and the symbiotic association affect establishment probability in different ways, and (2) a trade-off between the mean colony growth rates of the host alone and the symbiotic association could result in the evolution of either a symbiotic or parasitic relationship, based on a host decision.

SOCPROG programs: analysing animal social structures

SOCPROG is a set of programs which analyses data on animal associations. Data usually come from observations of the social behaviour of individually identifiable animals. Associations among animals, sampling periods, restrictions on the data and association indices can be defined very flexibly. SOCPROG can analyse data sets including 1,000 or more individuals. Association matrices are displayed using sociograms, principal coordinates analysis, multidimensional scaling and cluster analyses. Permutation tests, Mantel and related tests and matrix correlation methods examine hypotheses about preferred associations among individuals and classes of individual. Weighted network statistics are calculated and can be tested against null hypotheses. Temporal analyses include displays of lagged association rates (rates of reassociation following an association). Models can be fitted to lagged association rates. Multiple association measures, including measures produced by other programs such as genetic or range use data, may be analysed using Mantel tests and principal components analysis. SOCPROG also performs mark-recapture population analyses and movement analyses. SOCPROG is written in the programming language MATLAB and may be downloaded free from the World Wide Web.

Histone N-terminal Tails Interfere with Nucleosome Traversal by RNA Polymerase II

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.

Paenibacillus provencensis sp. nov., isolated from human cerebrospinal fluid, and Paenibacillus urinalis sp. nov., isolated from human urine

Gram-negative, spore-forming rods were isolated from a human urine sample (strain 5402403(T)) and a human cerebrospinal fluid sample (strain 4401170(T)). Based on genotypic characteristics, these strains belonged to the genus Paenibacillus and were closely related. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that they clustered with Paenibacillus massiliensis 2301065(T) (95.9 and 94.3 % 16S rRNA gene sequence similarity, respectively, for strains 5402403(T) and 4401170(T)), Paenibacillus illinoisensis NRRL NRS-61356(T) (90.6 and 93.8 %), Paenibacillus xylanilyticus XIL14(T) (95.3 and 95.4 %), Paenibacillus barcinonensis BP-23(T) (94.3 and 94.0 %), Paenibacillus pabuli NCIMB 12781(T) (89.1 and 92.3 %) and Paenibacillus amylolyticus NRRL NRS-290(T) (94.2 and 93.8 %). The predominant fatty acids were 15 : 0 anteiso (49.0 and 55.3 %, respectively, for strains 5402403(T) and 4401170(T)), 16 : 0 iso (15.4 and 13.5 %), 16 : 0 (7.6 and 3.6 %), 15 : 0 (6.3 and 2.8 %), 17 : 0 anteiso (5.7 and 7.5 %), 14 : 0 iso (4.1 and 2.7 %) and 15 : 0 iso (4.1 and 3.4 %). 16S rRNA gene sequence similarity between strain 4401170(T) and strain 5402403(T) was 98.4 %, but the DNA-DNA reassociation rate between the two strains was 53.2 %. So, considering the recommendations of the ad hoc committee, they do not belong to the same species. On the basis of phenotypic data and genotypic inference, it is proposed that the strains should be assigned to the novel species Paenibacillus urinalis sp. nov. (type strain 5402403(T) =CIP 109357(T) =CCUG 53521(T)) and Paenibacillus provencensis sp. nov. (type strain 4401170(T) =CIP 109358(T) =CCUG 53519(T)).

Substrate Enhances the Sensitivity of Type I Protein Kinase A to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.

Synthesis, structure, and activity of enhanced initiators for olefin metathesis.

A series of ruthenium olefin metathesis catalysts of the general structure (H(2)IMes)(PR(3))(Cl)(2)Ru=CHPh (H(2)IMes = 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene) have been prepared; these complexes are readily accessible in two steps from commercially available (H(2)IMes)(PCy(3))(Cl)(2)Ru=CHPh. Their phosphine dissociation rate constants (k(1)), relative rates of phosphine reassociation, and relative reaction rates in ring-opening metathesis polymerization (ROMP) and ring-closing metathesis (RCM) have been investigated. The rates of phosphine dissociation (initiation) from these complexes increase with decreasing phosphine donor strength. Complexes containing a triarylphosphine exhibit dramatically improved initiation relative to (H(2)IMes)(PCy(3))(Cl)(2)Ru=CHPh. Conversely, phosphine reassociation shows no direct correlation with phosphine electronics. In general, increased phosphine dissociation leads to faster olefin metathesis reaction rates, which is of direct significance to both organic and polymer metathesis processes.

Rate of endothelial expansion is controlled by cell:cell adhesion

Procedures used to alleviate blood vessel occlusion result in varying degrees of damage to the vascular wall and endothelial denudation. The presence of intact, functioning endothelium is thought to be important in controlling smooth muscle cell growth, and limiting the intimal thickening which results from damage to the vessel wall. Recovery of the endothelium is commonly slow and incomplete, due in part to endothelial lateral cell:cell adhesion, which limits cell migration and proliferation. We have investigated the effect of fibroblast growth factor 2 and vascular/endothelial growth factor on the relationship between the temporal distribution of the junctional adhesion proteins, platelet/endothelial cell adhesion molecule, vascular/endothelial cadherin and plakoglobin, and cellular migration and proliferation in an in vitro model of endothelial expansion. We found that whereas cell:cell junctions were initially disturbed to similar extents by single applications of the growth factors, outward cell migration and proliferation rates were inversely correlated with the speed at which cell:cell junctions were re-established. This occurred very rapidly with vascular/endothelial growth factor treatment and more slowly with fibroblast growth factor-2, resulting in more extensive outward migration and proliferation in response to the latter. Platelet/endothelial cell adhesion molecule and vascular/endothelial cadherin appeared to be associated with cell:cell junctional control of migration and proliferation, while plakoglobin did not contribute. It was concluded that the rate of endothelial expansion in response to growth factors, is limited by the rate of re-association of junctional complexes following initial disruption.

Capillary electrophoresis as a method to study DNA reassociation.

To develop analytical methodology to assess the genetic complexity of a DNA sample, capillary electrophoresis with laser-induced fluorescence detection is used to monitor the annealing process of DNA samples. Coated columns are filled with an entangled polymer solution shown to optimally separate DNA through size-selective capillary electrophoresis. DNA samples are denatured by heating in a boiling water bath for approximately 10 min and then cooled to approximately 25 degrees C below the melting point of the DNA sample to initiate the reassociation process. The DNA is detected by means of the laser-induced fluorescence of intercalated ethidium bromide, which produces a substantially greater signal for double- versus single-stranded DNA. The rate of reassociation is dependent upon the rate at which complimentary strands of DNA encounter each other and the degree of repeating base sequences in the sample (hence, the diversity of the DNA). Experimental parameters also influence the reassociation rate. The effects of salt concentration and incubation temperature are presented. Traditional plots of C(o)t (C(o) = DNA concentration and t = reassociation time) versus % recovery of double-stranded DNA signal are generated for PhiX 174 Hae III digest and 50 bp stepladder DNA, individually and combined, to calculate the reassociation rate constants for these samples. Because reassociation of individual fragments is observed by the CE-LIF method, more information about the samples is available than with less specific and time-consuming traditional methods of investigating DNA reassociation.

Burkholderia uboniae sp. nov., L-arabinose-assimilating but different from Burkholderia thailandensis and Burkholderia vietnamiensis.

A polar multitrichous gram-negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA-DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, bio-chemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA-DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone-8(Q8). G+C content of DNA is 69.71%.

Pathogenicity of vibrio splendidus strains associated with turbot larvae, scophthalmus maximus

Turbot larvae were challenged with eight strains of Vibrio splendidus isolated from diseased larvae, plus a ninth strain pathogenic to scallop larvae (A515; Nicolas et al. 1996). Six strains caused heavy mortality but the scallop pathogen and the other two strains did not. All the strains shared a large number of phenotypic traits, and an attempt was made to relate virulence to genotype and phenotype. Five of the six pathogenic strains were very similar, as shown by RAPD fingerprinting and phenotypic characteristics. The relatedness of the other strains was intermediate between the main pathogenic group and V. splendidus ATCC 33125, but the DNA-DNA homology between the pathogenic group and the reference strain was still high (78% of reassociation rate). The non-pathogenic isolates may be a useful tool for determining the possible virulence factors, as all the isolates differed by few characteristics.

Broad-scale analysis of soil microbial community DNA from Upland grasslands.

We have applied a broad-scale approach to the analysis of DNA extracted from soils which support characteristic grasslands at an upland site in the UK. To test for the degree of coherence between microbial and vascular communities, grasslands were characterised as 'improved', 'semi-improved', or 'unimproved', according to the degree of management they had received and consequent botanical composition. Microbial DNA was extracted directly from the grassland soils and analysed by three techniques: (i) thermal denaturation, which profiles the guanine and cytosine (G + C) base distribution within the community; (ii) cross hybridisation of the DNA which measures the degree of similarity between the samples; (iii) measurement of reassociation kinetics of denatured DNA, which provides a measure of the complexity of the DNA. Thermal denaturation revealed significant differences in the %G + C composition of the communities. DNA from the improved soil had the highest median %G + C value, whilst that from the unimproved soil had the lowest. The relative distribution of G + C bases also differed significantly between the samples from the three grasslands. Cross hybridisation of DNA from the different soils also indicated significant differences in the degree of similarity between the DNA from the grasslands, with unimproved showing 59% similarity to improved. Indices from the cross hybridisation assay suggested that, in terms of complexity, the samples ranked unimproved > semi-improved > improved. Reassociation kinetics supported this conclusion, but the rates of reassociation were such that less than 40% reassociation occurred over a 31-day period, thus preventing calculation of C(o)t1/2.

Mechanisms of modulation of voltage-dependent calcium channels by G proteins.

Voltage-dependent calcium channels (VDCCs) are key components in the complex functioning of excitable cells. Because of this, their regulation is of paramount importance to the control of cellular activity. VDCCs consist of a pore-forming α1 subunit, which has four domains each containing six putative transmembrane segments. From purification studies, these are associated with an intracellular β subunit. They also co-purify with an α2 subunit, which is entirely extracellular, linked into the membrane by S-S bonding to a transmembrane δ subunit (Witcher et al. 1993; Liu, De Waard, Scott, Gurnett, Lennon & Campbell, 1996) (Fig. 1). A number of different α1 subunits have been cloned; α1C, D and S all form 1,4-dihydropyridine (DHP)-sensitive L-type calcium channels, whereas α1A, B and E form P/Q-, N- and possibly R- or T-type channels, respectively. There are several means by which these channels may be modulated, but for neuronal channels, particularly N and P/Q, a major mechanism involves inhibitory modulation via the activation of heterotrimeric G proteins by seven transmembrane (7TM) receptors (for review see Dolphin, 1995). The key features of this inhibition are that it is always partial and is typified by a slowing of the current activation kinetics, which is thought to be due to a time-dependent recovery from voltage-dependent inhibition (Bean, 1989). The voltage dependence is manifested by a shift to more depolarized potentials of the current activation-voltage relationship, and the loss of inhibition at large depolarizations (Bean, 1989). There may also be additional mechanisms that are not voltage dependent, manifested by an incomplete ability of a depolarizing prepulse to reverse the inhibition (Diverse-Pierluissi & Dunlap, 1993), and the continuing presence of inhibition at large depolarizations measured from the tail current amplitude. Figure 1 The VDCC oligomeric complex Calcium channels are present in many tissues, where they fulfil a number of different specialized roles. In neurons the distribution of the channels is non-uniform, with α1B and α1A being particularly concentrated at synaptic terminals (Westenbroek, Hell, Warner, Dubel, Snutch & Catterall, 1992; Westenbroek et al. 1995). G protein-mediated modulation of these channels has been shown to occur at presynaptic terminals (Toth, Bindokas, Bleakman, Colmers & Miller, 1993). Evidence suggests that this mechanism may be responsible for at least some of the presynaptic inhibition of synaptic transmission mediated by a wide variety of 7TM receptors in many areas of the nervous system (Man-Son-Hing, Zoran, Lukowiak & Haydon, 1989; Hille, 1992; Toth et al. 1993; Dolphin, 1995). Activation of such receptors will reduce calcium entry into presynaptic terminals via VDCCs, but the effect should be frequency dependent. Inhibition will be reduced during a high frequency train as a result of the voltage dependence of the inhibitory modulation, providing a gain-setting mechanism. Relief of inhibition of calcium currents, evoked by action potential-like voltage waveforms, has been reported during high frequency trains (Williams, Serafin, Muhlethaler & Bernheim, 1997), and might contribute to the modulation of presynaptic inhibition depending on input frequency. The search for the molecular mechanism of this modulation is hotting up. Nevertheless, it should not be forgotten that indirect mechanisms such as presynaptic hyperpolarization, or direct mechanisms involving inhibition of exocytosis, are also likely to play a role in the modulation of presynaptic release of transmitter (Man-Son-Hing et al. 1989).

Refolding and reassociation of glycerol dehydrogenase from Bacillus stearothermophilus in the absence and presence of GroEL.

The refolding of the tetrameric, metalloenzyme glycerol dehydrogenase (GDH) from Bacillus stearothermophilus has been investigated using stopped-flow fluorescence and circular dichroism spectroscopy. The effects of metal ions on the refolding of the native enzyme and the refolding of a monomeric mutant ([A208]GDH) have also been studied. The refolding process of the wild-type enzyme is at least biphasic; 70% of the respective signal changes occur in the first 2 ms followed by a slower process with a half-life of 3 s. The presence of the metal ion does not affect the slowest biphasic refolding rate, which is virtually the same for all three versions of the enzyme. The presence of GroEL slows down the first phase of refolding. The reassociation of subunits was examined by measuring the regain in catalytic activity and the enhancement in the fluorescence emission from NADH on binding to the oligomeric form of the enzyme. The rate and extent of reassociation is dependent on enzyme concentration and the extent of reactivation is dependent on the presence of the metal ion. The reassociation process was more efficient in the presence of NADH particularly for the metal-depleted enzyme (apo-GDH). The presence of GroEL or GroEL plus ATP leads to a higher yield of reassociation and therefore catalytically active enzyme. The additional presence of Mg-ATP does not affect the extent of reassociation, but has a small positive effect on the rate of reassociation. These data suggest that GDH is bound weakly to GroEL and that GroES is not required for release of the protein.