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Biomedical subjects

G Rialdi

Publications and source records attributed to G Rialdi.

At least 19 recordsLinked to original sources
PubMed1999

Analytical titration curves of glycosyl hydrolase Cel45 by combined isoelectric focusing-electrophoresis.

The electrical charge of endocellulase Cel45-core has been determined by combined isoelectric focusing-electrophoresis in the range of pH 3-9. In order to transform electrophoretic mobility to absolute electrical charge value, several corrections were applied: the frictional coefficient theoretically calculated from the molecular dimensions depends on porous gel structure and on the ionic strength of the solution. By comparing the curve calculated according to the Linderstrom-Lang equation, the number of charged electrical groups exposed to the solvent and their apparent ionization constants, pK(o)i, can be determined. Furthermore, the macromolecule structure can be assumed not to change in this pH range. This finding is necessary to understand the structure and the electrical properties of the entire Cel45 molecule.

Aspergillus↗
PubMed1994

Decoupling of melting domains in immobilized ribonuclease A.

Ribonuclease A has been immobilized on silica beads through glutaraldeyde-mediated chemical coupling in order to improve the stability of the protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared with those of the free enzyme. The parameters describing the binding of the inhibitor 3'-CMP (Ka and delta H) as monitored by spectrophotometry and calorimetry were not significantly affected after immobilization. Conversely both the stability and unfolding mechanism drastically changed. Thermodynamic analysis of the DSC data suggests that uncoupling of protein domains has occurred as a consequence of the immobilization. The two state approximation of the protein unfolding process is not longer valid for the immobilized RNase. Protein stability strongly depends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobilization on a highly hydrophobic surface, the enzyme is partially in the unfolded state. The binding of a ligand is able to reorganize the protein structure into a native-like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3'-CMP.

Animals↗
PubMed1991

Differential scanning calorimetry characterization of rabbit brain membrane fractions.

Various membrane fractions have been prepared from rabbit brain synaptosomes by centrifugation on discontinuous sucrose gradients after osmotic shock. These fractions were characterized by electron microscopy (E.M.), SDS-PAGE and GABA binding. The fractions were then studied by differential scanning calorimetry (DSC). The calorimetric results indicate that all the fractions studied show thermal transitions at around 60 degrees C which correspond to the "melting" of membrane structures. An additional transition at higher temperature (82 degrees C) seems to be associated with an enrichment in central myelin fragments.

Animals↗
PubMed1990

Kinetics and thermodynamic study on the chemical reduction of macroimmunoglobulins.

The chemical reduction of human macroglobulin from its native pentameric form to reduced monomers has been followed using three different methods. The following parameters were measured under different conditions of pH, T and concentration: time dependent kinetics of the turbidity effect at 320 nm, chemical titration of -SH groups, and the determination of the heat effects evolved with time. The results obtained showed the same kinetics parameters for the reaction and indicate that the reduction of the 26 S-S bridges proceeds cooperatively from the pentameric structure to the monomeric one without the formation of intermediate states. The enthalpic contribution measured calorimetrically suggests that a conformational change of the chains structure occurs during the reduction.

Calorimetry↗
PubMed1985

Physico-chemical and thermodynamic properties of monomeric concanavalin A.

An alkylated monomer of Concanavalin A was prepared photochemically according to the method of Tanaka et al. (1981). Its affinities for methyl-alpha-D-gluco, methyl-alpha-D-manno and p-nitrophenyl-alpha-D-manno pyranoside were calculated. The enthalpies of these binding reactions were measured calorimetrically and the thermodynamic parameters were calculated. The values obtained suggest that the structure of the monomer differs from that of the dimeric and tetrameric molecules. Calorimetric studies also showed that the monomeric derivative reacts with IgM but not IgG. The enthalpy per binding site in the monomer-IgM reaction is equal to that of the monomer-mannose derivative reaction; mannose is the terminal residue of the saccharide chains of the IgM molecule. The stoichiometry of the reaction is ten ConA-m per IgM molecule.

Amino Acids↗
PubMed1985

Calorimetric analysis of phagocytosis of staphylococcal protein-A/IgG complexes.

We have analyzed calorimetrically the phagocytosis of the pseudoimmunecomplexes formed by staphylococcal protein A in presence of serum. Strong thermal effects were recorded during phagocytosis. They appear to be the sum of two distinct heat contributions. We have previously shown that these are the expression of two different non-mitochondrial O2 reduction pathways operating during the metabolic "burst" (Eftimiadi and Rialdi, 1982). Experiments carried out at different protein A/IgG ratios produced quantitatively and qualitatively different power-time curves. The data reported here indicate that although the immunoglobulins are essential for the triggering complex to form, the presence of complement is necessary to obtain maximal granulocytic metabolic activation.

Antigen-Antibody Complex↗
PubMed1985

A new calorimetric technique to analyze bacteria-phagocyte interactions.

We have assessed the use of a batch calorimeter in the analysis of bacteria phagocyte interactions. Results obtained using different strains of staphylococci agree with our previously reported flow calorimetric data. Phagocytosis of Staphylococcus aureus produces a greater metabolic activation energy expenditure with respect to other "saprophytic" staphylococci. The results obtained indicate that the batch calorimeter better fulfills all the requirements for routine phagocytosis assay.

Calorimetry↗
PubMed1984

Calorimetric study of rheumatoid factor binding to human IgG.

The binding of human IgM monoclonal rheumatoid factor (RF) to native human IgG was studied in a flow calorimeter. Thermal titration gave a enthalpy of binding of 5.5 X 10(4) J/mole site for the reaction, with 1 mole of RF binding to 2.5 moles of IgG; this corresponds to one RF subunit per IgG heavy chain. Similar experiments performed with heat-aggregated human IgG gave almost identical results. If the IgG molecule undergoes a conformational change upon heating, this is not such as to affect the enthalpic contribution of binding.

Antigen-Antibody Reactions↗
PubMed1984

The use of flow calorimetry in antigen-antibody reactions.

The binding of anti-IgM IgG to human monoclonal IgM has been studied calorimetrically and results have been compared with parallel turbidity measurements of the same reaction. In the first series of experiments using the flow calorimeter equipped with a mixing cell, varied flow rates gave identical values for the thermic contribution, indicating that the same quantity of heat was evolved in the different time intervals in which the reaction mixture was in the calorimetric cell. In the second set of experiments a flow-through cell was used. Reactants were mixed outside the calorimeter and no heat was detected upon entry of the pre-mixed reactants into the calorimetric cell. Results suggest that heat is evolved instantaneously upon mixing and no heat is produced during aggregation. Antibody-antigen binding can be followed in the absence of precipitation, as may occur in reactions involving monoclonal antibodies.

Animals↗
PubMed1984

Increased energy expenditure by granulocytes during phagocytosis of Staphylococcus aureus compared with other staphylococci.

The heat produced by human neutrophils during phagocytosis of different strains of Staphylococcus was analyzed by a flow calorimetric technique. At the same bacteria/leukocyte ratio Staphylococcus aureus yielded heat effects about twice that of other saprophytic staphylococci tested. In parallel experiments the degree of phagocytosis of radiolabeled bacteria was evaluated: S. aureus strains were the least phagocytosed. These data indicate that, for a single phagocytosed bacterium, S. aureus strains yield heat effects three to four times greater than do the saprophytic staphylococci tested (P less than .001). Previous studies have shown that the heat produced by activated neutrophils derives essentially from the aerobic catabolism of glucose involved in the production of bactericidal O2 metabolites and is directly proportional to O2 consumption. The results reported here indicate that less efficient internalization of a staphylococcal strain is correlated with a greater energy expenditure for O2 metabolite formation during the metabolic burst.

Calorimetry↗
PubMed1982

Calorimetric study of the binding reaction of concanavalin A with immunoglublins.

The thermal effects associated with the binding reaction of concanavalin A and immunoglobulins have been measured by calorimetry. IgG solutions do not generate heat on mixing with concanavalin A, confirming the low reactivity of IgG for the lectin molecule. IgM solutions, on the other hand, show a substantial enthalpic contribution of delta H = -24 +/- 1kJ/site for the binding reaction between the polysaccharide chains of human IgM macroglobulin and concanavalin. A stoichiometry is 10 monovalent concanavalin A molecules per intact macroglobulin and two bivalent concanavalin A molecules per two sites on the heavy chain of the reduced macroglobulin subunit.

Calorimetry↗
PubMed1981

Calorimetric study of manganese binding to concanavalin A.

The reaction of concanavalin A with Mn2+ has been studied calorimetrically. The binding enthalpy was measured at two different temperatures, 25 and 30 degrees C, in 10(-3) M acetate buffer; it was found to be constant between pH 4.0 and 5.0, delta H250 = 95 kJ/mol and delta H300 = 65 kJ/mol, respectively. The two S1 binding sites are identical and independent. Experiments at pH 5.6 are distorted by the heat of aggregation, which is several times higher than the heat of binding. Aggregation was demonstrated by spectrophotometric experiments and by light scattering. The presence of Mn2+ increases the stability of the protein molecule.

Binding Sites↗
PubMed1981

Calorimetric study of concanavalin A binding to saccharides.

The binding of concanavalin A in the dimer form to various saccharides has been studied by calorimetry, and estimates of the binding enthalpy and binding constants have been calculated. Methyl alpha-D-mannoside and methyl alpha-D-glucoside have a -- delta H0 of 21.5 and 11.5 kJ/mol, respectively, at both pH 4 and 4.5. The p-nitrophenyl derivatives react with enthalpic values of 15.6 and 14.6 kJ/mol. The galactosepyranosides show no heat effects during mixing with the protein solutions. The apparent binding enthalpies calculated from the variations of the equilibrium constants with temperature are in good agreement with the values measured experimentally. The two binding sites of the dimer form of concanavalin A are equal and independent, and the low enthalpies obtained do not justify a large conformational change during the reaction. The binding reaction has also been estimated for other sugars normally contained in glycoproteins.

Binding Sites↗