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M Coletta

Publications and source records attributed to M Coletta.

At least 127 records · Page 7Linked to original sources
PubMed1985

Evidence for two oxygen-linked binding sites for polyanions in dromedary hemoglobin.

The functional properties of dromedary hemoglobin have been studied as a function of chloride, polyphosphates and pH and compared with those of human hemoglobin. The two proteins have the same amino acid residues at the anion-binding sites as well as at the level of the groups responsible for the alkaline Bohr effect. Analysis of the experimental data reveals that: (a) intrinsic oxygen affinity and the Bohr effect are very similar for the two proteins; (b) the association equilibrium constants of chloride are substantially higher in the dromedary system, both in the unligated and ligated state; (c) two polyanion-binding sites occur in dromedary oxy and deoxyhemoglobin; (d) association constants of polyphosphates for the higher-affinity binding site (probably in the cavity between beta chains) are comparable for the two proteins under physiological conditions; association constants for the second binding site in dromedary hemoglobin are not affected by pH changes; (e) the dependence of oxygen affinity in dromedary hemoglobin upon chloride concentration is complex, this anion at relatively low concentrations reverses the effect of millimolar polyphosphate; (f) both in stopped-flow and flash photolysis experiments the kinetic behaviour of dromedary hemoglobin is consistent with the equilibrium results. The pronounced sensitivity to solvent composition of the functional properties of dromedary hemoglobin even in the oxy state stresses the potential relevance of this conformation for regulating the oxygen transport in vivo.

2,3-Diphosphoglycerate↗
PubMed1985

Kinetics of carbon monoxide binding to monomeric hemoproteins. Role of the proximal histidine.

The effect of pH on (i) the second-order rate constant for CO binding and (ii) the spectral properties of the deoxygenated derivative of several monomeric hemoproteins has been investigated in the pH range between 2.3 and 9.0. As in the case of 3-[1-imidazolyl]-propylamide monomethyl ester mesoheme, the rate constant for CO binding to sperm whale, horse, Dermochelys coriacea, Coryphaena hippurus, and Aplysia limacina myoglobins (the latter only in the presence of acetate/acetic acid mixture) increases, as the pH is lowered, to a value at least 1 order of magnitude higher than at pH 7.0. Such an effect is not observed in A. limacina myoglobin (in the absence of the acetate/acetic acid mixture) and Chironomus thummi thummi erythrocruorin. Moreover, the absorption spectrum, in the visible region, of the deoxy derivative of all these monomeric hemoproteins (with the exception of A. limacina myoglobin in the absence of the acetate/acetic acid mixture) undergoes a transition as the pH is lowered, an effect observed previously with 3-[1-imidazolyl]-propylamide monomethyl ester protoheme. On the basis of analogous spectroscopic and kinetic properties of chelated heme model compounds we attribute this behavior to the protonation of the N epsilon of the proximal imidazole involved in the bond with the iron atom. On the basis of this model the movement of the iron atom to the heme plane appears as a crucial step for CO binding, the activation free energy of the process amounting to approximately 2 kcal/mol.

Animals↗
PubMed1985

Emergence of TPA-resistant 'satellite' cells during muscle histogenesis of human limb.

Human satellite cells, obtained by surgical biopsies of traumatized legs of healthy individuals, were grown in culture in the presence of different concentrations of the phorbol ester tetradecanoyl-phorbol 12 acetate (TPA). Satellite cells, after an initial duplicative period, fused into large multinucleated myotubes which readily synthesized myosin and acetylcholine receptor (AChR). The presence of TPA at concentrations up to 10(-7) M did not affect the differentiation pattern, while higher concentrations were toxic. Thus human satellite cells are capable of differentiating in the presence of phorbol esters which block differentiation of embryonic myoblasts [1]. We then examined the appearance of TPA-resistant cells during human muscle histogenesis, since we had observed that differentiation of human myoblasts from a 6-week-old limb was completely and reversibly inhibited by 10(-7) M TPA. Differentiation of myoblasts from 6-, 7- and 8-week-old fetuses was completely inhibited by TPA. Myoblasts from 10-week-old limbs did not form myotubes in the presence of TPA; however, immunohistochemical staining with an antimyosin antibody revealed the presence of a few mononucleated myosin-positive cells which escaped the TPA-induced block of differentiation. At 12 weeks of development, a few oligonucleated, myosin-positive myotubes developed in cultures treated with TPA, and the level of AChR expressed (measured as [125I] alpha-bungarotoxin bound) reached 20% of controls. At 14 weeks of development, about half of the cells in culture were TPA-resistant and by 16 weeks of development no major differences could be detected between control and treated cells. We conclude from these data that a population of TPA-resistant myogenic cells emerges between the 10th and 14th week of human limb development and suggest that this population represents satellite cells.

Adolescent↗
PubMed1983

Reactivity of ferrous heme proteins at low pH.

The pH rate profile of sperm whale myoglobin (Mb) reacting with CO has been confirmed to follow the behavior previously reported (Giacometti, G.M., Traylor, T.G., Ascenzi, P., Brunori, M., and Antonini, E. (1977) J. Biol. Chem. 252, 7447-7448), and appears to be different from that obtained by others. The pH investigation has been extended to the CO-binding rates of Chironomus thummi thummi erythrocruorin and Aplysia limacina Mb, whose pH rate profile is different from that of sperm whale Mb. Besides the "base dissociation" mechanism previously invoked, the iron atom-heme plane distance is discussed as a possible determinant of the CO reactivity in these monomeric heme proteins.

Animals↗
PubMed1983

The reaction of trout hemoglobins with isocyanides.

Binding of alkylisocyanides of different bulkiness to the two major components of trout hemolysate is presented. In the case of trout hemoglobin I isocyanide binding is pH-independent, similar to O2 and CO, and the bulkiness of the ligand is related to the endothermicity of ligand binding to the T quaternary state. On the other hand, in trout hemoglobin IV the size of the ligand seems to affect the pH dependence of affinity and cooperativity. A comparison with other ligands, like O2, allows us to hint at possible stereochemical determinants of ligand binding in these two hemoglobins.

Animals↗
PubMed1983

[Microspectrophotometry in the study of physiological and pathological events in erythrocytes].

Microspectrophotometry turns out to be the ideal method for an accurate study of single cells, namely of red blood cells, from the biochemical and physiological standpoint. Hereafter several possible applications are reported: a. the kinetics of gas-exchange (O2 and CO) in human erythrocytes has been made possible by the photosensitivity of the Hb-CO complex. In this way, it has been possible to establish that both ligands recombine with intraerythrocyte hemoglobin following a zero-order kinetic process. This suggested that diffusion of a ligand across an unstirred layer of buffer (approximately 5.10 mu thick) all around the cell could be the rate-limiting step of this recombination. Moreover, an intracellular facilitated oxygen diffusion has been observed and possible physiological implications have been shortly mentioned; b. the kinetics of intracellular polymerization of sickle cell hemoglobin in single red blood cells has been measured by quickly (approximately 1 msec) flashing-off the CO employing a cw Argon ion laser. The polymerization of deoxyhemoglobin has been followed by detecting the increased light scattering of the laser beam itself. The distribution of delay times (td) in several patients has been found significantly correlated to the severity of the disease, supporting the hypothesis that the td is a determinant factor in the pathophysiology of the disease. Possible therapeutic applications of this method are briefly discussed; c. the intraerythrocyte distribution of different hemoglobins of trout (Salmo irideus) among several erythrocytes has been observed by means of different physiochemical properties of single components. These turned out to be homogeneously present in all red blood cells studied.

Animals↗
PubMed1982

Kinetics of sickle haemoglobin polymerization in single red cells.

Kinetic studies on solutions of purified haemoglobin S indicate that the rate of intracellular polymerization is an important variable in the pathophysiology of sickle cell disease. Until now, however, no experimental technique has been available to measure directly the kinetics of intracellular polymerization. Indirect methods, which use visual determination of cellular shape changes or changes in filterability of red cell suspensions, have given apparently conflicting results. Here we report our initial results on the application of a laser-photolysis, light scattering technique to measure directly the kinetics of haemoglobin S polymerization in single red cells. In our experiment, deoxyhaemoglobin S is rapidly formed by photolysing the carbon monoxide complex with an argon ion laser focused inside the cell, and the change in scattered light is used to detect the appearance of polymer. We find a very wide distribution of delay times, ranging from 1 ms to greater than 100 s, indicating that the polymerization inside red cells proceeds by the same nucleation and growth mechanism as in solutions of purified haemoglobin S.

Erythrocytes↗
PubMed1980

Root effect of Panulirus interruptus hemocyanin.

Panulirus interruptus hemocyanin exhibits a progressive decrease in oxygen affinity and a parallel loss of cooperativity with decrease in pH, resulting in an apparent loss of the oxygen-binding capacity of the protein. For a characterization of this system, oxygen-binding curves have been determined over the complete range of oxygen saturation, applying a special technique which involves high-pressure spectrophotometry. Although the oxygen-binding behavior as a function of pH is complex and cannot be described within the frame of a simple two-state Monod-Wyman-Changeux model, the observed Root effect is clearly related to a progressive stabilization of a low oxygen affinity state of the protein and functional heterogeneity is not apparent.

Animals↗
PubMed1980

In vitro differentiation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells.

Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2--3 days, followed by a shorter phase (1--2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [3H]thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [3H]thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase (CPK) specific activity is observed. Satellite cells are able to recognize co-cultured embryonic myoblasts ([3H]thymidine-labelled): hybrid myotubes containing labelled and unlabelled nuclei are formed in these experimental conditions. Satellite cells from dystrophic animals are able to differentiate in culture and do not show appreciable differences as compared to their normal counterparts. In dystrophic myotubes, however, CPK specific activity is almost twice that observed in normal myotubes. Human dystrophic satellite cells from biopsies of adult muscle cultured in similar conditions grow and fuse into multinucleated myotubes showing a behaviour identical to normal controls.

Animals↗
PubMed1978

A comparative approach to protein- and ligand-dependence of the Root effect for fish haemoglobins.

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

Amino Acid Sequence↗
PubMed1978

A macromolecular transducer as illustrated by trout hemoglobin IV.

Oxygen binding by trout Hb IV has been investigated as a function of pH up to 10 atmospheres (1 MPa) of pure O2. The results bring out an extreme proton-oxygen linkage, which gives rise to a Root effect. They are discussed in relation to the function of the hemoglobin as an oxygen pump. The system is of special interest as providing a prototype of a macromolecule acting as a transducer by coupling two allosterically linked reactions.

Allosteric Regulation↗