Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry

The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.     

Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects

Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose-1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the method of high-sensitivity differential scanning calorimetry (HS-DSC). The dependence of the denaturation temperature on the heating rate reveals in the case of both proteins a non-equilibrium character of the denaturation transition in applied conditions. Developed kinetic approach allows the determination of an equilibrium transition temperature as well as the rate constants of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conformational stability of KTI within pH range from 2.0 to 11.0. It allowed the determination of the pH dependencies: of the excess free energy of denaturation, of the activation enthalpy and entropy of denaturation as well as of the denaturation rate constant. Conclusions have been made suggesting the contribution of side-chain hydrogen bonds in the stabilisation of the native and activated states of KTI.     

The role of copper in the stability of ascorbate oxidase towards denaturing agents

The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.     

Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability

Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.     

Two-state irreversible thermal denaturation of Euphorbia characias latex amine oxidase

Thermal denaturation of Euphorbia latex amine oxidase (ELAO) has been studied by enzymatic activity, circular dichroism and differential scanning calorimetry. Thermal denaturation of ELAO is shown to be an irreversible process. Checking the validity of two-state it really describes satisfactorily the thermal denaturation of ELAO. Based on this model we obtain the activation energy, parameter T(*) (the absolute temperature at which the rate constant of denaturation is equal to 1 min(-1)), and total enthalpy of ELAO denaturation. HPLC experiments show that the thermal denatured enzyme conserves its dimeric state. The N(2)-->kD(2) model for thermal denaturation of ELAO is proposed: where N(2) and D(2) are the native and denatured dimer, respectively.     

Global changes of chlorophyll excitonic interactions in photosystem II during thermal denaturation

Photosystem II (PSII) is a multisubunit chlorophyll-binding enzyme that absorbs light to catalyze water oxidation and plastoquinone reduction. Chlorophyll excitonic interaction changes in PSII were studied by absorption and circular dichroism spectra from 25 degrees C to 80 degrees C, and protein subunit denaturation was monitored by differential scanning calorimetry. A four-stage process of chlorophyll excitonic interaction change was observed being correlated with the denaturation of protein subunits.     

Alteration of thermal stability of glucose oxidase associated with the redox states

By the method of differential scanning calorimetry, it was found that thermal stability of glucose oxidase was dependent on its redox states. The oxidized form showed an apparent denaturation temperature at 76°C and the denaturation enthalpy was approximately 865 kcal/mol. On reduction of the enzyme, the denaturation temperature increased by about 10°, but no significant change was seen in the denaturation enthalpy. The activation energies of the denaturation of the oxidized and the reduced enzymes were about 89 and 103 kcal/mol, respectively. These results may imply conformational changes in the catalytic turnover of this enzyme.     

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T ^*, temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.     

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins. Correspondence to: J. M. Sanchez-Ruiz     

The Kinetic Stability of Cytochrome c Oxidase: Effect of Bound Phospholipid and Dimerization

Thermally induced transitions of the 13-subunit integral membrane protein bovine cytochrome c oxidase (CcO) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). Thermal denaturation of dodecyl maltoside solubilized CcO proceeds in two consecutive, irreversible, kinetically driven steps with the apparent transition temperatures at ∼ 51°C and ∼ 61°C (5μM CcO at scan rate of 1.5 K/min). The thermal denaturation data were analyzed according to the Lyubarev and Kurganov model of two consecutive irreversible steps. However, because of the limitation of the model to describe the complex mechanism of the thermal denaturation of CcO, the obtained results were utilized only for comparison purposes of kinetic stabilities of CcO under specific protein concentration (5μM) and scan rate (1.5 K/min). This enabled us to show that both the amphiphilic environment and the self-association state of CcO affect its kinetic stability. Kinetic stabilities of both steps are significantly decreased when all of the phospholipids are removed from CcO by phospholipase A₂ (the half-life decreases at 37°C). Conversely, dimerization of CcO induced by sodium cholate significantly increases its kinetic stability of only the first step (the half-life increases at 37°C). Protein concentration-dependent nonspecific oligomerization also indicate mild stabilization of CcO. Both, reversed-phase high-performance liquid chromatography (HPLC) and SDS-PAGE subunit analysis reveal that the first step of thermal denaturation involves dissociation of subunits III, VIa, VIb, and VIIa, whereas the second step is less well defined and most likely involves global unfold and aggregation of the remaining subunits. Electron transport activity of CcO decreases in a sigmoidal manner during the first transition and this dependence is very well described by kinetic parameters for the first step of the thermal transition. Therefore, dissociation of subunit III and/or VIIa is responsible for temperature-induced inactivation of CcO because VIa and VIb can be removed from CcO without affecting the enzyme activity. These results demonstrate an important role of tightly bound phospholipids and oligomeric state (particularly the dimeric form) of CcO for kinetic stability of the protein.     

Differential scanning calorimetry of asparate transcarbamoylase and its isolate subunits.

The thermal denaturation of aspartate transcarbamoylas of Escherichia coli was investigated by differential scanning calorimetry. Isolated regulatory and catalytic subunits were heat denatured at 55 and 80 degrees C, respectively. In contrast, the intact enzyme was denatured in two steps. A small endotherm near 73 degrees C was assoicated with denaturation of the regulatory subunits and the major endotherm at 82 degrees C with denaturation of the catalytic subunits. Thus regulatory subunits are stabilized against heat denaturation by more than 17 degrees C when incorporated in the enzyme. Similar conclusions were obtained from measurements of the enthalpy of heat denaturation. Regulatory subunits yielded a much lower value of the enthalpy of denaturation, 1.91 cal/g, than that found for the catalytic subunit, 3.94 cal/g, or typical globular proteins (4 to 6 cal/g). When the regulatory subunits were incorporated into aspartate transcarbamoylase their enthalpy of denaturation was increased 125% (to 4.3 cal/g). The enthalpy of the catalytic subunits in the intact enzyme was increased 38% (enthalpy of denaturation of 5.43 cal/g). Stabilization of the isolated catalytic subunit as well as the intact enzyme was achieved by the addition of the bisubstrate analog N-(phosphonacetyl)-L-aspartate. Similarly the allosteric effectors, CTP and ATP, stabilized the isolated regulatory subunits or those subunits within the intact enzyme. However, the addition of the bisubstrate analog caused a decrease in the enthalpy of denaturation of the regulatory subunits within the enzyme. These results are consistent with other studies of the ligand-promoted conformational changes in the native enzyme.     

Differential scanning calorimetric study of the thermal denaturation of aspartate transcarbamoylase of Escherichia coli

The thermal denaturation of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of high-sensitivity differential scanning calorimetry (DSC). As previously reported [Vickers, K.P., Donovan, J.W., & Schachman, H.K. (1978) J. Biol. Chem. 253, 8493-8498], the denaturational endotherm consists of two peaks, the lower of which is due to denaturation of the three regulatory, r2, subunits while the upper involves the two catalytic, c3, subunits. The temperature of maximal excess apparent specific heat, tm, of the lower peak is raised from the value of 51.4 degrees C for the isolated subunit to 66.8 degrees C as a result of subunit interactions, whereas tm for the c3 peak is essentially the same in the isolated subunit and in the holoenzyme, indicating that the denatured r2 subunits do not interact with the c3 subunits. The total specific denaturational enthalpy for c6r6, 4.83 +/- 0.16 cal g-1, is significantly larger than the weighted mean, 4.08 cal g-1, of the enthalpies for c3 and r2. The fact that no endotherm is observed when previously scanned protein is rescanned indicates that the denaturation is irreversible, as is also the case with the r2 and c3 subunits. Empirical justification for analyzing the data in terms of equilibrium thermodynamics is cited. The observed DSC curves can be expressed within experimental uncertainty as the sum of five sequential two-state steps. The value of t 1/2, the temperature of half-completion, for each step increases with increasing protein concentration, indicating that some dissociation of the protein takes place during denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides

The thermal unfolding of the catabolite activator protein (CAP) of Escherichia coli and the complexes it forms with adenosine cyclic 3',5'-phosphate (cAMP) and guanosine cyclic 3',5'-phosphate (cGMP) was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of CAP at pH 7.00 gave an irreversible, symmetrical denaturation curve with a single peak. Distinctly different, more complex DSC curves were obtained for the thermal denaturation of the cAMP-protein and cGMP-protein complexes. The DSC data indicate intermolecular cooperation among CAP dimers, with the extent of oligomerization remaining unchanged during unfolding of the protein. The DSC curves for the thermal denaturation of the cAMP-protein complex and cGMP-protein complex have been resolved into three and two components, respectively, according to the model of independent two-state processes. Analysis of the DSC data suggests two and three independent domains for cGMP-protein and cAMP-protein complexes, respectively, with dissociation of mononucleotide occurring in the second component in both cases during protein denaturation. Furthermore, our studies indicate that the presence of either ligand alters the degree of oligomerization of CAP dimers, cAMP having a greater effect than cGMP.     

Thermal inactivation of electron-transport functions and F0F1-ATPase activities

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains.

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.     

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).     

The thermal unfolding and domain structure of Na+/K+-exchanging ATPase. A scanning calorimetry study.

The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC). The excess heat capacity function of Na+/K+-ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the beta subunit, while two other domains belong to the alpha subunit. The thermal unfolding of the low-temperature domain leads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximately 12 J.g-1), suggesting that thermal denaturation of Na+/K+-ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.