Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC

Escherichia coli RecBCD is a highly processive DNA helicase involved in double-strand break repair and recombination that possesses two helicase/translocase subunits with opposite translocation directionality (RecB (3′ to 5′) and RecD (5′ to 3′)). RecBCD has been shown to melt out ∼ 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg²⁺-dependent, but ATP-independent reaction. Here, we examine the binding of E. coli RecBC helicase (minus RecD), also a processive helicase, to duplex DNA ends in the presence and in the absence of Mg²⁺ in order to determine if RecBC can also melt a duplex DNA end in the absence of ATP. Equilibrium binding of RecBC to DNA substrates with ends possessing pre-formed 3′ and/or 5′ single-stranded (ss)-(dT)_n flanking regions (tails) (n ranging from zero to 20 nt) was examined by competition with a fluorescently labeled reference DNA and by isothermal titration calorimetry. The presence of Mg²⁺ enhances the affinity of RecBC for DNA ends possessing 3′ or 5′-(dT)_n ssDNA tails with n < 6 nt, with the relative enhancement decreasing as n increases from zero to six nt. No effect of Mg²⁺ was observed for either the binding constant or the enthalpy of binding (ΔH_obs) for RecBC binding to DNA with ssDNA tail lengths, n ≥ 6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg²⁺, at least 4 bp at the duplex end become accessible to KMnO₄ attack, consistent with melting of the duplex end. Since Mg²⁺ has no effect on the affinity or binding enthalpy of RecBC for a DNA end that is fully pre-melted, this suggests that the role of Mg²⁺ is to overcome a kinetic barrier to melting of the DNA by RecBC and presumably also by RecBCD. These data also provide an accurate estimate (ΔH_obs = 8 ± 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC.     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

Molecular recognition of single-stranded RNA: Neomycin binding to poly(A)

Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single-stranded oligonucleotide (A₃₀) with a K_d in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47 °C. The poly(A) duplex, which melts at 44 °C (pH 5.5), was observed to melt at 61 °C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.     

Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K_d = 3.62 ± 2.1 × 10⁻⁸ M) or the RNA corresponding sequence (K_d = 2.7 ± 0.82 × 10⁻⁸ M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.     

Structure-specific binding of [Co(phen)2(HPIP)] to a DNA duplex containing sheared G:A mismatch base pairs

The binding of a Co(III) complex to the decanucleotide d(CCGAATGAGG)₂ containing two pairs of G:A mismatches was studied by 2D-NMR, UV absorption, and molecular modeling. NMR investigations indicate that racemic [Co(phen)₂(HPIP)]Cl₃ [HPIP = 2-(2-hydroxyphenyl) imidazo [4,5-f][1,10] phenanthroline] binds the decanucleotide by intercalation: the HPIP ligand selectively inserts between the stacked bases from the minor groove at the terminal regions and from the major groove at the sheared region. Further, molecular modeling revealed that the recognition shows strong enantioselectivity: the Λ-isomer preferentially intercalates into the T₆G₇:A₅A₄ region from the DNA major groove, while Δ-isomer favors the terminal C₁C₂:G₁₀G₉ region and intercalates from the minor groove. Detailed energy analysis suggests that the steric interaction, especially the electrostatic effect, is the primary determinants of the recognition event. Melting experiments indicate that binding stabilizes the DNA duplex and increases the melting temperature by 9.5 °C. The intrinsic binding constant of the complex to the mismatched duplex was determined to be 3.5 × 105 M⁻¹.     

A novel intramolecular G-quartet-containing fold of single-stranded d(GT)8 and d(GT)16 oligonucleotides

Human genome is shown to be enriched with (GT)_n stretches of lengths from 8 to 20 dinucleotides. Low temperature (T ≤ 10 °C) conformations of d(GT)_n oligonucleotides (n = 7, 8, 12, 16, 20) were studied by means of circular dichroism (CD), thermal melting, ethidium bromide (EtBr) probing and single nucleotide substitutions. Rotational relaxation times for EtBr:d(GT)_n complexes confirmed a monomolecular state of the oligonucleotides. CD spectra indicated involvement of all guanines of d(GT)₈ and d(GT)₁₆ in G-quartets, while dT(GT)₇, d(GT)₁₂ and d(GT)₂₀ were shown to be only partially ordered. The schemes of the d(GT)₈ and d(GT)₁₆ folds are suggested.     

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is ∼ 250 Å, with ∼ 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing ∼ 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.     

Effects of summer- and winter-like acclimation on the thermoregulatory behavior of fed and fasted desert hamsters, Phodopus roborovskii

1.

Thermoregulatory behavior of fed and fasted desert hamsters (Phodopus roborovskii) acclimated to summer- [16 light (L):8 dark (D), ambient temperature (T_a)=26.5 °C] and winter-like (8L:16D, T_a=10 °C) conditions was studied. Body temperature (T_b), selected temperature and activity were measured in hamsters placed in a thermal gradient system for 48 h.     

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.     

Seasonal changes in the thermal tolerances of the toad Rhinella arenarum (Bufonidae) in the Monte Desert of Argentina

We studied the thermal tolerances of Rhinella arenarum during the dry and wet seasons of the Monte Desert in San Juan Province, Argentina. This toad had differences in CT_max between dry and wet seasons, and the CT_max values were higher in the wet season (Austral summer). Operative temperature, body temperature, environmental maximal temperature, and relative humidity were related to CT_max, suggesting seasonal acclimatization of R. arenarum. Additionally, the CT_max recorded for R. arenarum was 36.2 °C, and the maximum ambient temperature recorded during the toads' activity time was 37 °C. Also, the CT_min recorded for R. arenarum was 5.3 °C and the minimum environmental temperature recorded was 7.2 °C. The wide thermal tolerance range recorded and the relationship between tolerance limits and the environmental extremes indicate that seasonal acclimatization is an effective mechanism by which toads can raise their thermal tolerance, allowing them to survive in the challenging conditions of the Monte Desert. Additional studies are needed to understand the relationship between the thermal tolerance of this desert amphibian and the environmental parameters that influence its thermal physiology.     

Thermogenic characteristics and evaporative water loss in the tree shrew (Tupaia belangeri)

Thermogenic characteristics and evaporative water loss were measured at different temperatures in Tupaia belangeri. The thermal neutral zone (TNZ) of T. belangeri was 30–35 °C. Mean body temperature was 39.76±0.27 °C and mean body mass was 100.86±9.09 g. Basal metabolic rate (BMR) was 1.38±0.03 ml O₂/g h. Average minimum thermal conductance (C_m) was 0.13±0.01 ml O₂/g h °C. Evaporative water loss in T. belangeri increased when the temperature rose; the maximal evaporative water loss was 3.88±0.41 mg H₂O/g h at 37.5 °C. The results may reflect features of small mammals in the sub-tropical plateau region: T. belangeri had high basal metabolic rate and high total thermal conductance, compared with the predicted values based on their body mass whilst their body temperatures are relatively high; T. belangeri has high levels of evaporative water loss and poor water-retention capacity. Evaporative water loss plays an important role in temperature regulation.     

Studies on the interaction of copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate with calf thymus DNA

The interaction of copper complexes of (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) with calf thymus DNA (ct-DNA) was investigated by UV-visible (UV-Vis), fluorescence and circular dichroism along with melting studies. It was observed that both copper complexes quench the fluorescence intensity of ethidium bromide bound ct-DNA upon binding, resulting in a ground state complex formation by a static quenching process. The binding constants evaluated from fluorescence data were supported by the UV-Vis study. The values ranged from 0.84 to 1.07 × 10⁵ M⁻¹ and 1.14 to 1.04 × 10⁵ M⁻¹ for Cu(II)-ECG and Cu(II)-EGCG, respectively for the temperature range 21-42 °C with two binding sites. Thermodynamic parameters obtained are suggestive of the involvement of different modes of interaction during binding for each complex although both were found to be intercalating in nature. Circular dichroism studies and variations in the melting temperature reveal unwinding of the ct-DNA helix with conformational changes due to binding.     

High Temperature Stabilization of DNA in Complexes with Cationic Lipids

The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100°C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T_m(labile), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T_m(stable), at 105 to 115°C. The parameter T_m(stable) did not depend on the charge ratio, R(±). Instead, the amount of thermostable DNA and the enthalpy ratio ΔH_(stable)/ΔH_(labile) depended upon R(±) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(±) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(±) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130°C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.     

Activation of transgene expression in skeletal muscle by focused ultrasound

To correlate thermal dose from focused ultrasound (FUS) with gene expression and tissue injury, a temperature plateau strategy was employed. Plasmids encoding luciferase gene under the control of hsp70B promoter were transfected into the right gastrocnemius muscle in a rat via electroporation. One day after transfection, hind limbs were treated with 3.3-MHz focused ultrasound, using one of four different temperature plateaus with spatial-peak time-average focal temperatures (T_SPTA) of 46 °C, 48 °C, 51 °C and 62 °C. The treatment duration at the plateau temperature was varied from 0 to 30 s. Gene expression was analyzed in vivo one day following FUS treatment, and H&E staining was employed to assess tissue injury. Gene activation and tissue damage correlated closely with thermal dose. The highest level of gene activation was induced by FUS at T_SPTA = 51 °C for 20 s, which was found to be statistically equivalent to that produced by water-bath hyperthermia.     

Interindividual differences in thermal comfort and the responses to skin cooling in young women

The present study aims to understand the effects of interindividual differences in thermal comfort on the relationship between the preferred temperature and the thermoregulatory responses to ambient cooling. Thirteen young women subjects chose the preferred ambient temperature (preferred T_a) in a climate chamber and were categorized into the H group (preferring ≥29 °C; n=6) and the M group (preferring <29 °C; n=7). The H group preferred warmer sensations than the M group (P<0.05) and the average of preferred T_a was 27.6 °C and 30.2 °C in the M group and H group, respectively. Then all subjects were exposed to temperature variations in the climate chamber. During T_a variations from 33 °C to 25 °C, the H group felt colder than the M group, although no difference was noted in the T_sk (mean skin temperature) and T_s-hand between the 2 groups. From the view of the relationship between the T_sk and thermal sensation, although the thermal sensitivity to the T_sk was almost similar in the H and M groups, the H group might have lower threshold to decreasing T_a than the M group.     

Red to near-infrared electrophosphorescence from an iridium complex coordinated with 2-phenylpyridine and 8-hydroxyquinoline

An iridium complex coordinated with 2-phenylpyridine (ppy) and 8-hydroxyquinoline (q), ppy₂Irq, was synthesized and its thermal stability, absorption, photoluminescence, crystal structure and electrophosphorescence were characterized. The melting point of this material reaches as high as 374 °C and does not suffer decomposition upon heating at high vacuum therefore can be well sublimated. When ppy₂Irq was used as a guest emitting material in the electrophosphorescent device, the emission is 100% saturated red light starting at ∼600 nm, extending into the near-infrared region. The bathochromic shift, compared to the fluorescence and phosphorescence from Alq₃, Ptq₂ and Ir(ppy)₃, was analyzed to originate from the triplet excited state of 8-hydroxyquinoline ligand and the crystal structure analysis excludes the origin of π-π intermolecular interactions.     

Deposition and characterization of ZnO thin films from a novel hexanuclear zinc precursor

The high nuclearity zinc complex, Zn₆(OAc)₈(μ-OH)₂(dmae)₂(dmaeH)₂ (1) (OAc = acetate and dmaeH = N,N′-dimethylaminoethanol), having a low decomposition temperature and sufficiently high solubility in non-polar solvents, was synthesized by a simple chemical technique in high yield and analyzed by melting point, elemental analysis, FTIR, NMR, single crystal X-ray crystallography and thermal analysis. Aerosol-assisted chemical vapor deposition technique was used to deposit a high-quality thin film with good adhesion to the glass substrate at relatively low temperature (320 °C). Scanning electron microscopy of the film shows clearly distinct crystallites of uniform shape with 2.4-2.9 μm size. Powder X-ray diffraction measurements have indicated the deposition of a crystalline phase of hexagonal ZnO with space group P63mc.     

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase

F₁-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F₁-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 10⁶ M⁻¹ s⁻¹ at 4°C to 4.3 × 10⁷ M⁻¹ s⁻¹ at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F₁-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.