Biomedical subjects

E Freire

Publications and source records attributed to E Freire.

At least 127 records · Page 7Linked to original sources

PubMed1989

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition.

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.

Calorimetry, Differential Scanning↗

PubMed1989

Aerobic training effects on maximum oxygen consumption, lactate threshold and lactate disappearance during exercise recovery of dogs.

1. Dogs were submitted to an aerobic training schedule and its maximum oxygen consumption, lactate threshold and lactate concentration during recovery were compared among the following conditions: not trained (UT), after 1 month of training (T1), after 2 months of training (T2) and after detraining (DT). 2. Maximum oxygen consumption increased significantly in relation to UT condition only at T2 condition. The detraining reversed this alteration. 3. Lactate threshold when expressed as Vo2 or absolute work load increased significantly after aerobic training (T2) but did not present any alteration when it was expressed as % of Vo2 max. 4. The lactate decreasing during recovery did not differ between the four experimental conditions (after 10 min). 5. The latency time for the lactate concentration to reach the top values was reduced by aerobic training (T2).

Animals↗

PubMed1989

Breathing pattern during pharmacological activation and blockade of the intermediate area of the ventrolateral surface of the cat medulla.

The present study analyzes the respiratory pattern of chloralose-(50-60 mg/kg, iv) anesthetized cats treated with Nembutal (NE) (30 mg/ml), glycine (GL) (200 mg/ml) or leptazol (LE) (200 mg/ml) topically applied to the intermediate area of the ventrolateral surface of the medulla oblongata in a volume of 20 microliters. Application of NE and GL produced a decrease in ventilation (approximately 24%) and tidal volume (approximately 25%) suggesting that the intermediate area facilitates respiratory drive and inhibits the inspiratory off-switch mechanism. These results are consistent with the view that intermediate area is necessary for the central chemosensitivity to CO2. The topical application of LE produced an increase in inspiration time (12.5%), expiration time (20.8%) and tidal volume (7%). The increased tidal volume caused by LE is compatible with its action as a GL antagonist.

Animals↗

PubMed1988

Combining thermostable mutations increases the stability of lambda repressor.

We have combined three mutations previously shown to stabilize lambda repressor against thermal denaturation. Two of these mutations are in helix 3, where Gly-46 and Gly-48 have been replaced by alanines [Hecht, M. H., et al. (1986) Proteins: Struct., Funct., Genet. 1, 43-46]. The other mutation, which replaces Tyr-88 with cysteine, allows the protein to form an intersubunit disulfide bond [Sauer, R. T., et al. (1986) Biochemistry 25, 5992-5998]. Calorimetric measurements show that the two alanine substitutions stabilize repressor by about 8 degrees C, that the disulfide bond stabilizes repressor by about 8 degrees C, and that the triple mutant is 16 degrees C more stable than wild-type repressor.

Amino Acid Sequence↗

PubMed1988

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction.

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.

Kinetics↗

PubMed1988

Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1.

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

PubMed1988

Frequency spectrum of enthalpy fluctuations associated with macromolecular transitions.

A multifrequency calorimeter has been designed to measure the amplitude and time regime of the enthalpic fluctuations associated with structural or conformational transitions in biological macromolecular systems. The heat capacity function at constant pressure is directly proportional to the magnitude of the enthalpic fluctuations in a system. Biological macromolecules undergo thermally induced transitions of different kinds. Within the transition region, these systems exhibit relatively large enthalpy fluctuations that give rise to the characteristic peaks observed by conventional differential scanning calorimetry. The multifrequency calorimeter developed in this laboratory has been designed to measure the frequency spectrum of the enthalpy fluctuations, thus allowing us to estimate thermodynamic parameters as well as relaxation times. This information is obtained from the attenuation in the amplitude or phase-angle shift of the response of the system to a periodic temperature oscillation. This instrument has been used to study the gel-liquid crystalline transition of phosphatidylcholine bilayers. The frequency-temperature response surface for large dimyristoyl phosphatidylcholine vesicles has been measured in the frequency range 0.04-1 Hz. The data are consistent with two enthalpic relaxation processes with time constants on the order of 3.8 s and 80 ms at the midpoint of the main gel-liquid crystalline transition.

Calorimetry, Differential Scanning↗

PubMed1987

Thermodynamic characterization of interactions between ornithine transcarbamylase leader peptide and phospholipid bilayer membranes.

The interactions of the targeting sequence of the mitochondrial enzyme ornithine transcarbamylase with phospholipid bilayers of different molecular compositions have been studied by high-sensitivity heating and cooling differential scanning calorimetry, high-sensitivity isothermal titration calorimetry, fluorescence spectroscopy, and electron microscopy. These studies indicate that the leader peptide interacts strongly with dipalmitoylphosphatidylcholine (DPPC) bilayer membranes containing small mole percents of the anionic phospholipids dipalmitoylphosphatidylglycerol (DPPG) or brain phosphatidylserine (brain PS) but not with pure phosphatidylcholines. For the first time, the energetics of the leader peptide-membrane interaction have been measured directly by using calorimetric techniques. At 20 degrees C, the association of the peptide with the membrane is exothermic and characterized by an association constant of 2.3 X 10(6) M-1 in the case of phosphatidylglycerol-containing and 0.35 X 10(6) M-1 in the case of phosphatidylserine-containing phospholipid bilayers. In both cases, the enthalpy of association is -60 kcal/mol of peptide. Additional experiments using fluorescence techniques suggest that the peptide does not penetrate deeply into the hydrophobic core of the membrane. The addition of the leader peptide to DPPC/DPPG (5:1) or DPPC/brain PS (5:1) small sonicated vesicles results in vesicle fusion. The fusion process is dependent on peptide concentration and is maximal at the phase transition temperature of the vesicles and minimal at temperatures below the phase transition.

Calorimetry, Differential Scanning↗

PubMed1987

Differential detergent solubility investigation of thermally induced transitions in cytochrome c oxidase.

The thermal denaturation of membrane-reconstituted cytochrome c oxidase (EC 1.9.3.1) occurs at approximately 63 degrees C as determined by high-sensitivity differential scanning calorimetry. The heat capacity profile associated with this process is characterized by the presence of two well-defined peaks, indicating that all the enzyme subunits do not have the same thermal stability. This thermal denaturation of the enzyme complex is coupled to a change in its solubility properties. This change in solubility allows separation of the native and denatured protein fractions by detergent solubilization followed by centrifugation under conditions in which only the native fraction is solubilized. Using this principle, it has been possible to study the denaturation of membrane-reconstituted cytochrome c oxidase and quantitatively identify the protein subunits undergoing thermal denaturation using computer-assisted gel electrophoresis analysis. This technique allows calculation of single-subunit thermal denaturation profiles within the intact enzyme complex, and as such, it can be used to obtain transition temperatures, molecular populations, and van't Hoff enthalpy changes for individual protein subunits, thus complementing results obtained by high-sensitivity differential scanning calorimetry.

Animals↗

PubMed1987

Thermodynamic and phase characterization of phosphatidylethanolamine and ganglioside GD1a mixtures.

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside GD1a had a gel-liquid crystalline phase transition between 15 and 25 degrees C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21 degrees C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4-16.2 mol%. The high enthalpy change (37 kcal/mol of GD1a added into the PE bilayer) indicates the existence of PE-GD1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-GD1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-GD1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside GD1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20-25 degrees C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to GD1a ratio at constant temperatures.

Calorimetry, Differential Scanning↗

PubMed1987

Kinetics of ganglioside transfer between liposomal and synaptosomal membranes.

The transfer of ganglioside GM1 from micelles to membranes and between different membrane populations has been examined by using a pyrene fatty acid derivative of the ganglioside. The transfer of gangliosides from micelles to membranes depends on the physical state as well as the molecular composition of the acceptor vesicles. At 30 degrees C, the transfer of micellar gangliosides to dipalmitoylphosphatidylcholine (DPPC) large unilameller vesicles (Tm = 41.3 degrees C) is characterized by a rate constant of 0.01 min-1; at 48 degrees C, however, the rate constant is 0.11 min-1. Below the phase transition temperature, the activation energy is 25 kcal/mol whereas above the phase transition it is 17 kcal/mol. Similar experiments performed with synaptic plasma membranes yielded a rate constant of 0.05 min-1 at 37 degrees C. The rate of transfer of ganglioside molecules, asymmetrically located on the outer layer of donor vesicles, to acceptor vesicles lacking ganglioside depends on the physical state of both the donor and acceptor vesicles. For the transfer of ganglioside from DPPC (donor) vesicles to dimyristoylphosphatidylcholine (DMPC) (acceptor) vesicles, the rates were essentially zero at 15 degrees C in which both vesicle populations were in the gel phase, 0.008 min-1 at 30 degrees C in which DPPC is in the gel phase and DMPC is in the fluid phase, and 0.031 min-1 at 48 degrees C in which both vesicle populations are in the fluid phase. The transfer of ganglioside from DPPC vesicles to synaptic plasma membranes was also dependent on the physical state of the donor vesicles and showed an inflection point at the phase transition temperature of DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Dynamic analysis of differential scanning calorimetry data.

The apparent heat capacity function measured by high-sensitivity differential scanning calorimetry contains dynamic components of two different origins: (1) an intrinsic component arising from the finite instrument time response; and (2) a sample component arising from the kinetics of the thermal transition under study. The intrinsic instrumental component is always present and its effect on the shape of the experimental curve depends on the magnitude of the calorimeter response time. Usually, high-sensitivity instruments exhibit characteristic time constants varying from 10 to 100 s. This slow response introduces distortions in the shape of the heat capacity function especially at fast scanning rates. In addition to this instrumental component, dynamic effects due to sample relaxation processes also contribute to the shape of the experimental heat capacity profile. Since the nature and magnitude of these effects are a function of the kinetic parameters of the transition, they can be used to obtain kinetic information. This communication presents a dynamic deconvolution technique directed to remove artificial distortions in the shape of the heat capacity function measured at any scanning rate, and to obtain a kinetic characterization of a thermally induced transition. The kinetic characterization obtained by this method allows the researcher to obtain transition relaxation times as a continuous function of temperature. This technique has been applied to the thermal unfolding of ribonuclease A and the pretransition of dipalmitoylphosphatidylcholine (DPPC). In both systems the transition relaxation times are temperature dependent. For the protein system the relaxation time is very slow below the transition temperature (approximately 30 s) and very fast above Tm (less than 1 s) in agreement with direct kinetic measurements. For the pretransition of DPPC, the relaxation time is maximal at the transition midpoint and of the order of approx. 40 s.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1986

Direct measurement of the energetics of association between myelin basic protein and phosphatidylserine vesicles.

A newly designed high-sensitivity isothermal reaction calorimetry system has been used to investigate the thermodynamics of the association between myelin basic protein and phosphatidylserine vesicles. This instrument has allowed us to measure directly the energetics of the protein-lipid interaction under various conditions. Above the phospholipid phase transition temperature the enthalpy of association is highly exothermic amounting to -160 kcal/mol of protein. Below the phospholipid phase transition temperature the enthalpy of association is exothermic at protein/lipid ratios smaller than 1/50 and endothermic at higher protein/lipid ratios. These studies indicate that the association of myelin basic protein to phosphatidylserine vesicles consists of at least two stages involving different types of binding. The first stage, at low protein/lipid ratios, involves a strong exothermic association of the protein to the membrane and the second, at high protein/lipid ratios, a weaker association probably involving attachment of the protein to the membrane surface only. In the gel phase the second binding stage is endothermic and appears to be correlated with the formation of large vesicle aggregates. This vesicle aggregation is a reversible process dependent upon the physical state of the membrane. The isothermal titration studies have been complemented with high-sensitivity differential scanning calorimetry experiments. It is shown that the dependence of the phospholipid transition enthalpy on the protein/lipid molar ratio can be expressed in terms of the different protein-membrane association enthalpies in the gel and fluid phases of the membrane.

Animals↗

PubMed1986

Thermotropic characterization of phosphatidylcholine vesicles containing ganglioside GM1 with homogeneous ceramide chain length.

The thermotropic behavior of dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine large unilamellar vesicles containing ganglioside GM1 of homogeneous long chain base composition has been studied by high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. At neutral pH and in the absence of Ca2+, the thermotropic behavior of these systems is independent of the ganglioside chain length composition. The presence of Ca2+ at concentrations higher than 5 mM induces ganglioside phase separation in a manner dependent upon the length difference between the ganglioside long chain base and the phosphatidylcholine acyl chains. The analysis of the chain length dependence of the thermotropic behavior suggests that the driving force for ganglioside phase separation is not a Ca2+-induced cross-bridging of the ganglioside head group but a passive ganglioside exclusion from Ca2+-perturbed phosphatidylcholine-rich regions within the bilayer. Experiments with native ganglioside GM1, primarily a mixture of C18:1 and C20:1 long chain bases, indicate that the individual components of the mixture maintain their characteristic behavior within the lipid bilayer matrix. These results, together with the presence of a phase transition in native GM1 micellar dispersions, absent in purified C18:1 or C20:1 ganglioside micelles, strengthen the idea of a possible role of chain length composition in the modulation of ganglioside function.

Animals↗

PubMed1986

Lateral diffusion of ganglioside GM1 in phospholipid bilayer membranes.

The lateral diffusion coefficient of ganglioside GM1 incorporated into preformed dimyristoylphosphatidylcholine (DMPC) vesicles has been investigated under a variety of conditions using the technique of fluorescence photobleaching recovery. For these studies the fluorescent probe 5-(((2-Carbohydrazino)methyl)thio)acetyl) amino eosin was covalently attached to the periodate-oxidized sialic acid residue of ganglioside GM1. This labeled ganglioside exhibited a behavior similar to that of the intact ganglioside, and was able to bind cholera toxin. The lateral diffusion coefficient of the ganglioside was dependent upon the gel-liquid crystalline transition of DMPC. Above Tm the lateral diffusion coefficient of the ganglioside was 4.7 X 10(-9) cm2 s-1 (with greater than 80% fluorescence recovery). This diffusion coefficient is significantly slower than the one previously observed for phospholipids in DMPC bilayers. The addition of increasing amounts of ganglioside, up to a maximum of 10 mol %, did not have a significant effect on the lateral diffusion coefficient or in the percent recovery. At 30 degrees C, the lateral mobility of ganglioside GM1 was not affected by the presence of 5 mM Ca2+, suggesting that, at least above Tm, Ca2+ does not induce a major perturbation in the lateral organization of the ganglioside molecules. The addition of stoichiometric amounts of cholera toxin to samples containing either 1 or 10 mol % ganglioside GM1 produced only a small decrease in the measured diffusion coefficient. The fluorescence recovery after photobleaching experiments were complemented with excimer formation experiments using pyrene-phosphatidylcholine. Above the transition temperature the presence of 10 mol % ganglioside GMI induced a large decrease in the rate of excimer formation. These results also indicated that the addition of ganglioside GMI to phospholipid bilayer vesicles induces a significant restriction in the lateral mobility parameters of the lipid bilayer and that the presence of Ca2' does not have a further effect in the mobility of the probe molecules.

Animals↗

PubMed1985

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)

Animals↗

PubMed1985

Calorimetric and fluorescence characterization of interactions between enkephalins and liposomal and synaptic plasma membranes containing gangliosides.

The interactions of the opioid peptide [D-Ala2]methionine-enkephalinamide with dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing gangliosides GM1, Gd1a, and Gt1b and synaptic plasma membranes selectively enriched with dimyristoylphosphatidylcholine (DMPC) and ganglioside Gd1a have been investigated by using high-sensitivity differential scanning calorimetry. In the absence of gangliosides, the addition of enkephalinamide in concentrations of up to 10(-3) M does not induce any appreciable change in the heat capacity function of DPPC. In the presence of gangliosides, however, changes in the heat capacity function were observed with as little as micromolar concentrations of the enkephalinamide; the same is true for DMPC-Gd1a-enriched synaptic membranes. The magnitude and the nature of the enkephalinamide effect depend on the type of ganglioside studied. For DPPC vesicles containing ganglioside GM1 only a slight broadening in the heat capacity function and a small upward shift in the transition temperature were observed. For DPPC vesicles containing ganglioside Gd1a the effect was more dramatic; enkephalinamide concentrations as low as 10(-5) M caused the appearance of two well-defined peaks in the heat capacity function in contrast to the one peak observed in the absence of enkephalinamide. In the case of DPPC vesicles containing ganglioside Gt1b the enkephalinamide effect was seen at concentrations of 10(-4) M or higher. Synaptic plasma membranes were isolated from bovine brain, selectively enriched with exogenous lipid, and their thermotropic behavior was characterized by steady-state fluorescence spectroscopy and differential scanning calorimetry. This lipid enrichment results in the appearance of a membrane phase transition otherwise absent in the intact membrane preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1.

The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane. In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy. In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer. Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer. On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles. These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗