Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital.

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.

Effect of the antitumour protein alpha-sarcin on the thermotropic behaviour of acid phospholipid vesicles.

The antitumour protein alpha-sarcin modifies the thermotropic behaviour of phospholipid vesicles. This has been studied by fluorescence depolarization measurements and differential scanning calorimetry. A surface protein-phospholipid interaction is detected by measuring the polarization degree of TMA-DPH-labelled vesicles. At the higher protein/lipid molar ratios studied, the alpha-sarcin-vesicles complexes exhibit different thermotropic behaviour depending on whether they are prepared above or below the Tm of the corresponding phospholipid. Labelling of the protein with photoactive phospholipids has also been considered. alpha-Sarcin penetrates the bilayer deep enough to be labelled with the photoactive group located at the C-12 of the fatty acid acyl chain of phospholipids forming vesicles.

Thermal unfolding of the cytotoxin alpha-sarcin: phospholipid binding induces destabilization of the protein structure.

The effect of membrane binding on the structure and stability of the cytotoxin alpha-sarcin has been studied by differential scanning calorimetry, Fourier-transform infrared and fluorescence spectroscopic techniques. The thermal unfolding of alpha-sarcin in aqueous solution fits into a two-state transition characterized by a transition temperature (Tm) of 52.6 degrees C and a calorimetric enthalpy (delta Hcal) of 136 kcal/mol. Upon interaction with phosphatidylglycerol vesicles, alpha-sarcin undergoes conformational changes, as deduced from the FTIR and fluorescence emission spectra. These changes result in a decreased Tm and delta Hcal values for the thermal unfolding of phospholipid-bound alpha-sarcin. The lower Tm value for lipid-bound alpha-sarcin is also observed at the level of secondary and tertiary structures, based on analyses of both the amide I' infrared spectrum and the tryptophan emission of the protein as a function of temperature, respectively. The results obtained indicate a protein destabilization promoted by the phospholipid interaction.

Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains.

BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent.

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.

Regulation of phosphorylase b by AMP. The effects of enzyme concentration.

The enthalpies of binding of AMP to phosphorylase b have been measured as a function of enzyme concentration in glycylglycine buffer, pH 6.9. The results show how a conformational transition, which takes place in the concentration range of 1.7 to 2.5 mg/ml of phosphorylase b, affects the enthalpies of the two binding sites per monomer for the allosteric activator AMP. The enthalpies of the AMP interaction with its higher and lower affinity binding sites are -220 and -640 kJ (mol monomer)-1 at an enzyme concentration of 1 mg/ml, and -120 and -360 kJ (mol monomer)-1 at 2.7 mg/ml. The conformational transition of phosphorylase b alters the reactivity of the slow -SH groups of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid), suggesting that the environment of these groups are affected by the process. On the other hand, kinetic data show that the saturation of both classes of AMP binding sites affects the catalytic behavior and that their specific effects on the catalytic process are altered by the enzymatic transition dependent on the enzyme concentration. Thus, a strong inhibition is associated with the saturation of the weaker affinity AMP binding sites at 3 mg/ml while the saturation of these weaker affinity binding sites at 1 mg/ml produces an important activation of the enzyme.

A model for the behaviour of phosphorylase b. The generation of different binding sites via intermediate enzymatic states.

The model given in this paper can be applied to enzymatic systems which have more than two conformational states in equilibrium and which clearly exhibit heterogeneity in the binding of one ligand. The model we propose makes possible quantitative interpretation of our experimental results and of those of many other workers as well. In some cases calorimetric, dialysis and kinetic magnitudes, when plotted against ligand concentration, give multiregional or "stepwise" curves. We suggest that such a behaviour arises because total occupation of one class of binding sites completely moves the enzyme towards a different conformational state in which the affinity for the ligand is greatly increased by the formation of a new class of binding sites. Our calorimetric results for the interaction between some nucleotides and phosphorylase b closely conform to our model.

AMP interaction sites in glycogen phosphorylase b. A thermodynamic analysis.

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.

Thermodynamics of Ca2+ binding to calmodulin and its tryptic fragments.

The binding of Ca2+ to calmodulin and its two tryptic fragments has been studied using microcalorimetry. The binding process is accompanied by the uptake or release of protons, depending on the ionic strength. With no added salt, the total enthalpy change for the binding of four calcium ions to calmodulin is -41 kJ mol-1 but in the presence of 0.15 mM KCl delta Htot is +17 kJ mol-1. The mode of binding of Ca2+ is also completely different with and without added salt. It is also shown that for the C-terminal fragment of calmodulin, TR2C, the drastic reduction in delta Gtot for the binding process on increasing the ionic strength is largely an enthalpic effect. Domain interactions in calmodulin are indicated by the fact that the sum of the enthalpies of calcium binding to the two tryptic fragments is not the same as the total binding enthalpy to calmodulin itself. The binding of Ca2+ to calmodulin has also been studied calorimetrically at different temperatures in the range 21-37 degrees C. delta Cp is large and negative in this interval.

Thermodynamics of nucleotides binding to phosphorylase b.

The effects of several chemical modifications in the AMP molecule on its interaction with phosphorylase b are examined by microcalorimetry, equilibrium dialysis, light scattering and ultracentrifuge experiments. In this work we report the results obtained for eight AMP analogues corresponding to different substituents in the puric base or in the ribose, or to different positions of the phosphate. The thermodynamic properties of the interaction between the phosphorylase b and the above mentioned nucleotides are also reported. The following conclusions were obtained: a) Except for IMP and 2'dIMP all the nucleotides studied clearly show two types of binding sites in the enzyme. b) The interaction of the nucleotide with its weaker affinity binding site is highly dependent upon chemical alterations in the puric base. c) Both the amino group in C(6) and the N(1) of the adenine in the AMP seem to play an important role in the conformational transitions induced by the nucleotide on the enzyme. d) The tetramerization of phosphorylase b in the presence of 10(-2) M AMP and in the conditions of the ultracentrifuge experiments is drastically affected by modifications in the ribose-phosphate part of the AMP molecule.

Design and testing of a new microcalorimetric vessel for use with living cellular systems and in titration experiments.

A new microcalorimetric vessel primarily intended for use with living cellular systems and in titration experiments has been designed and tested. The vessel, which forms a modular system, fits into an ampoule measuring cylinder of the LKB 'BioActivity Monitor'. It can be used with different sample cups, volume 1-3 ml, and can be equipped with different types of stirrer and sample holders for cellular materials. Experiments can be performed with or without medium perfusing through the vessel. Small quantities of reagents can be added to the sample compartment during the measurements. Stepwise calorimetric titrations can be performed by an automatic procedure. Test experiments reported include results of measurements with human T-lymphoma cells in stirred suspension and melanoma cells adhered to a polystyrene film in a stirred perfusing medium. Results from titration experiments where N-acetyl-D-alanine was bound to ristocetin A are reported, delta G degree' = -16.5 +/- 0.2 kJ mol-1 and delta H degree' = -32.1 +/- 0.4 kJ mol-1.

Thermochemistry of the interaction between peptides and vancomycin or ristocetin.

The thermodynamics of the interaction between the glycopeptide antibiotics vancomycin and ristocetin and bacterial peptidoglycan peptide analogs have been studied by means of a microcalorimetric titration technique. From results of the calorimetric measurements, changes in Gibbs energies, enthalpies, entropies and heat capacities for the binding reactions have been calculated. The derived thermodynamic data have been discussed on the basis of stereochemical data available for the interaction of acetyl-D-alanyl-D-alanine with each of the two antibiotics. The significance of entropic factors connected with conformational changes of the antibiotics is stressed.

Physical properties of membrane lipids isolated from a thermophilic eubacterium (Thermus sp.).

Membranes from a thermophilic eubacterium, Thermus sp. strain SPS 11, isolated from thermal springs of São Pedro do Sul spa (Portugal), are characterized for having two main polar lipids, a glycolipid (GL) with four monosaccharide residues, which at 73 degrees C accounts for 95% of the carbohydrate in the total lipid extracts, and a glycophospholipid (PL) which at 73 degrees C accounts for about 90% of the lipid phosphorous. A complex mixture of carotenoids (CA) makes up 11% by weight of the total membrane lipids. The branched fatty acyl chains (iso C15 and iso C17) comprise about 90% of the alifatic moieties of the polar lipids of this bacterium. Moreover, when the growth temperature increases from 50 to 73 degrees C there is an increase of the iso C17/ iso C15 ratio and of the GL/PL ratio. We have studied the biophysical properties of bilayers (as multilamellar liposomes) prepared from GL, PL and the mixtures of PL, GL and CA in proportions found in the membranes of bacteria growing at their optimal growth temperature, using polarization of DPH fluorescence, low and wide-angle X-ray diffraction and differential scanning calorimetry. The three techniques agree in showing the presence of a broad phase transition from a gel (L beta) phase to a liquid-crystal (L alpha) phase between 8 and 30 degrees C, for all the lipid dispersions studied except for the GL. Although all the dispersions studied form a bilayer structure at all the temperatures studied, only the mixture of the three components (PL, GL + CA) avoids the phase separation present in the mixtures of PL + CA at temperatures lower than 30 degrees C and PL + GL at temperatures lower than 55 degrees C. Our results are compared with those of Pinheiro et al. (1978) obtained with the 31p-NMR technique and applied to the study of the same samples.

Absceso cerebral causado por salmonella typhi: reporte de caso / Brain abscess caused by salmonella typhi: case report

Se presenta el caso de un paciente de 50 años de edad, quien es admitido a la emergencia de adultos, con cuadro un convulsivo asociado a fiebre de una semana de evolución. El estudio de tomografía cerebral reveló la presencia de imagen heterogénea en lóbulo frontal , se interviene quirúrgicamente con hallazgo de un absceso cerebral logrando el aislamiento de Salmonella typhi, cumple cuatro semanas de tratamiento con ceftriaxona intravenosa con mejoría tanto clínica como radiológica...(AU)

We present the case of a 50-year-old patient, who is admitted to the emergency of adults, with a convulsive symptoms associated with fever of one week of evolution. The cerebral tomography study revealed the presence of heterogeneous image in the frontal lobe, it was surgically intervened with the finding of a cerebral abscess achieving the isolation of Salmonella typhi, it was four weeks of treatment with intravenous ceftriaxone with clinical and radiological improvement ... (AU )

Interaction of beta-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies.

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

Analysis of the solvent effect on the photophysics properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN).

The absorption and emission spectroscopic properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) have been studied in a large number of protogenic, nonprotogenic, and amphiprotic solvents. The data obtained can be explained by the inclussion of a new term in the Lippert equation which takes into account the acidity of the solvent. This finding indicates that some precaution should be taken when using PRODAN as an indicator of the polarity of protein cavities if the environments involved include acid sites.

Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus.

The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.