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M Castagnola

Publications and source records attributed to M Castagnola.

At least 19 recordsLinked to original sources
PubMed2001

Role of ergothioneine on S-nitrosoglutathione catabolism.

Ergothioneine (ESH) is a low-molecular-mass thiol present in millimolar concentrations in a limited number of tissues, including erythrocytes, kidney, seminal fluid and liver; however, its biological function is still unclear. In the present study we investigated the role of ESH in the catabolism of S-nitrosoglutathione (GSNO). The results show that: (1) GSNO decomposition is strongly influenced by ESH (k"=0.178+/-0.032 M(-1) x s(-1)); (2) ammonia is the main nitrogen-containing compound generated by the reaction; and (3) nitrite is practically absent under both aerobic and anaerobic conditions. These findings are markedly different from those reported for the GSH-induced decomposition of GSNO, in which the nitrogen-containing end products are nitrite, ammonia and nitrous oxide (N(2)O) under aerobic conditions but nitrite, ammonia, nitric oxide (NO) and small quantities of hydroxylamine under anaerobic conditions. Considering the high concentration of ESH in specific cells, the reaction with GSNO should be considered as an important molecular event occurring in the cell.

Ergothioneine↗
PubMed2001

Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection.

A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, alpha-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.

Acids↗
PubMed2000

Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis.

The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.

Amino Acid Sequence↗
PubMed2000

Adult and fetal haemoglobin J-Sardegna [alpha50(CE8)His-->Asp]: functional and molecular modelling studies.

Haemoglobin (Hb) J-Sardegna [alpha50(CE8)His-->Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 degrees C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 degrees C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the alpha(1)-beta(1) interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [alpha50(CE8)His-->Arg] at the same position.

2,3-Diphosphoglycerate↗
PubMed2000

Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis.

A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.

Electrophoresis, Capillary↗
PubMed2000

Separation of diadenosine polyphosphates by capillary electrophoresis.

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.

Borates↗
PubMed2000

Purification and properties of a nucleotide pyrophosphatase from lentil seedlings.

A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 +/- 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca2+, Mg2+, and Mn2+, whereas Cu2+, Zn2+, and Ni2+ ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.

Binding Sites↗
PubMed2000

Blood bank conditions and RBCs: the progressive loss of metabolic modulation.

BACKGROUND: Human RBC metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of storage on this metabolic modulation and its relationship with the alteration of membrane protein composition. STUDY DESIGN AND METHODS: RBCs stored in CPD-saline-adenine-glucose-mannitol were assayed for glucose uptake and partition between glycolysis and the pentose phosphate pathway at high and low oxygen saturation by nuclear magnetic resonance spectroscopy after 1, 14, 21, 35, and 42 days of storage. Membrane protein composition was determined by SDS-PAGE on Days 1, 14, 35, and 42. Metabolic values and 2,3 DPG concentration were also measured after rejuvenation for 1 hour at 37 degrees C with pyruvate-inosine-phosphate-adenine solution on Day 21. RESULTS: Metabolic differences between RBCs incubated at high and low oxygen saturation decreased during storage, and, on Day 35, the two groups did not have significant differences (p = 0.111). SDS-PAGE showed that membrane protein composition was concurrently modified. The percentage of unmodified band 3 decreased during storage, principally between Days 14 and 35. In rejuvenated RBCs, oxygen-dependent modulation was not restored. CONCLUSIONS: RBCs stored in CPD-saline-adenine-glucose-mannitol do show a progressive loss of oxygen-dependent metabolic modulation, which is not restored after rejuvenation and which seems partly related to modifications in membrane proteins, mainly band 3.

Adenine↗
PubMed1999

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.

Erythrocytes↗
PubMed1999

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Journal Article↗
PubMed1999

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.

Adaptation, Physiological↗
PubMed1998

Circe's haemoglobins, pig-human hybrids: functional characterization and structural considerations.

We report the isolation and the functional characterization of alpha and beta chains from pig (Sus scropha domesticus) haemoglobin, as well as of the pig-human hybrid haemoglobins, alpha2(h)beta2(p) and alpha2(p)beta2(h) (i.e. Circe's haemoglobins), obtained by mixing the purified alpha and beta pig chains respectively with the corresponding partner human chains. Their functional properties have been compared with those of both parental haemoglobins in order to obtain information on the role of the different subunits and of their inter-relationships, both at the structural and functional levels. The results indicate that the functional properties of both hybrids are closer to those of the parental haemoglobin that provides the beta chains, confirming the major role of the beta chains in determining the oxygen affinity and the modulation mechanisms of the tetrameric molecule. This is supported by the thermodynamic properties, since the very low DeltaH of oxygen binding that characterizes pig haemoglobin and the alpha2(h)beta2(p) hybrid haemoglobin may be taken as the reflection of specific structural properties of pig beta chain.

Amino Acids↗
PubMed1998

Thermodynamics of inositol hexakisphosphate interaction with human oxyhemoglobin.

The interaction of inositol hexakisphosphate (IHP) with oxygenated human adult hemoglobin (Hb) was investigated at 25 degreesC. The affinity of IHP for oxygenated Hb is strongly pH-dependent, and potentiometric measurements of proton uptake and release upon IHP addition have shown that over the range between pH 8.0 and pH 6.0 in oxygenated Hb there are three groups of residues that change their pKa values after IHP addition, likely because of their interaction with negative charges of the heterotropic effector. On the basis of previous calculations on the electrostatic properties of human Hb (Matthew, J. B., Hanania, G. I. H., and Gurd, F. R. N. (1979) Biochemistry 18, 1919-1928; Lee, A. W.-m., Karplus, M., Poyart, C., and Bursaux, E. (1988) Biochemistry 27, 1285-1301), two of these groups might be Val1beta and His143beta, which are located in the beta1beta2 dyad axis, where they have been also proposed to interact with 2,3-diphosphoglycerate, whereas the third group does not appear easily identifiable. Calorimetric measurements of the heat associated with IHP binding at different pH values over the same range indicate that IHP binding is mostly enthalpy-driven at pH < 7 and mostly entropy-driven at pH > 7.

Adult↗
PubMed1998

Glycated human hemoglobin (HbA1c): functional characteristics and molecular modeling studies.

A minor hemoglobin component of human red blood cell hemolysate, HbA1c, is the result of the non-enzymatic reaction of glucose with the alpha-amino groups of the valine residues at the N-terminus of the beta-chains of human hemoglobin. In this paper, the effect of protons, chloride and 2,3-diphosphoglycerate (DPG) on the functional properties of HbA1c has been investigated in some details. Moreover, the structural modifications induced on the native molecule by the sugar moieties, studied by computer modeling, do agree with the observed functional alterations. In particular, the functional results indicate that: (a) the low-affinity conformation (or T-state) of HbA1c is destabilized by the chemical modification per se; (b) the Bohr effect is reduced with respect to that of native HbA0; (c) the affinity of the T-state of HbA1c for 2,3-diphosphoglycerate is about 2.6 x lower than that of the corresponding conformational state of HbA0, while the R-state is less affected with, the affinity being 1.7 x lower. At the structural level, computer modeling studies show that the two sugar moieties are asymmetrically disposed within the 2,3-diphosphoglycerate binding site. In addition, molecular mechanics and dynamics calculations concerning the interaction with 2,3-diphosphoglycerate indicate that while in HbA0 the effector can assume two different stable orientations, in glycated Hb only one orientation is possible. All together, the results show that glycation of the Val 1 residues of both beta-chains does not impair the binding of DPG but imposes a different mode of binding by changing the internal geometry of the complex and the surface distribution of the positive electrostatic potential within the binding pocket.

Chemical Phenomena↗
PubMed1998

Predictive model for capillary electrophoretic peptide mobility in 2,2,2-trifluoroethanol-water solution.

Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2-trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00-4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge-to-size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00-2.25), either in water or a TFE-water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.

Electrophoresis, Capillary↗
PubMed1998

The pH dependence of predictive models relating electrophoretic mobility to peptide chemico-physical properties in capillary zone electrophoresis.

We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8).

Amino Acid Sequence↗
PubMed1997

Peptide analysis by capillary (zone) electrophoresis.

In this review various aspects concerning the application of capillary (zone) electrophoresis for peptide analysis will be critically examined. First, the basic instrumental requirements of CE apparatus and the strategies employed to enhance sensitivity in the analysis of underivatized sample are described. Multidimensional separative techniques of complex peptide mixtures that use CE as final step and the coupling of CE with mass spectrometry are subsequently discussed. A theoretical section describes the relationships existing between peptide mobility and the pH of the separation buffer. These relationships evidence that proton dissociation constants and Stokes radius at different protonation stages can be calculated by measuring the electrophoretic mobility at different pH values. Investigation of peptide mobility dependence on pH allows us to establish the optimum conditions, in terms of resolution, for peptide separation. Subsequently, a critical discussion about semiempirical models predicting peptide mobility as a function of chemico-physical peptide properties is presented. A section is devoted to the description of principles of peptide affinity capillary electrophoresis, underlining the similarity with peptide-proton interaction. CE separations performed in aquo-organic solvents are also critically discussed, showing the good performance obtained by using water-2,2,2-trifluoroethanol solutions. Finally, selected CE applications for the determination of peptide chemico-physical properties and conventional analysis, like peptide mapping, are reported.

Buffers↗