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Biomedical subjects

E Antonini

Publications and source records attributed to E Antonini.

At least 37 records · Page 2Linked to original sources
PubMed1983

Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs.

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.

Animals↗
PubMed1983

Kinetic studies on cytochrome c oxidase inserted into liposomal vesicles. Effect of ionophores.

Cytochrome c oxidase from ox heart was inserted into artificial liposomal vesicles obtained by sonication of purified soya-bean phospholipids. The cytochrome oxidase vesicles showed a respiratory control ratio of about 2. Spectroscopic properties in the visible and Soret regions and kinetics of CO binding are similar to those of the soluble oxidase. The catalytic efficiency of the cytochrome oxidase vesicles in oxidizing cytochrome c increases as a result of the formation of the 'pulsed' form of the oxidase and of the presence in the reaction mixture of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and nonactin. Analysis of the experimental results obtained under several conditions supports the conclusions that: (i) the alkalinization of the internal microenvironment in the liposomal vesicle is not by itself responsible for the decrease in catalytic activity; (ii) the electrical potential difference created during turnover by proton consumption and/or pumping through the liposome wall is an important mechanism of control in the chain of events leading to the oxidation of external cytochrome c.

Anti-Bacterial Agents↗
PubMed1983

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study.

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.

Animals↗
PubMed1983

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of Panulirus interruptus hemocyanin.

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.

Journal Article↗
PubMed1983

Studies of the antimicrobial activity of ovotransferrin.

The antibacterial activity of ovotransferrin (conalbumin) against different bacterial species was studied in vitro. The most sensitive species were Pseudomonas sp., E. coli, S. mutans; and the most resistant ones S. aureus, Proteus sp., Klebsiella. The bacteriostatic activity of conalbumin in various conditions was also tested. The presence of bicarbonate ions always increased the activity of conalbumin, while an antagonistic effect of citrate was observed in bacteria with a receptor for the iron-citrate complex. Experiments with conalbumin covalently linked to Sepharose 4B indicated that its antibacterial activity may not be due simply to the removal of iron from the medium, but probably involves other metals and an interaction with the bacterial surface. The in vitro studies carried out with conalbumin and lactoferrin demonstrated that the two proteins produced a similar inhibition of growth of E. coli and S. mutans. The in vivo studies showed that the protective effect of conalbumin in newborn guinea pigs with E. coli by gastrointestinal route was similar to that naturally provided by the lactoferrin present in milk.

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

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin.

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.

Apoproteins↗
PubMed1982

Structural heterogeneity and subunit composition of horse ferritins.

Structural, spectroscopic, and immunological properties of horse ferritins extracted from spleen, liver, and heart were studied to test the hypothesis that the different tissue ferritins are hybrids composed of variable proportions of two subunit types. The weight-average molecular weights determined by sedimentation velocity and gel filtration increase from 460 000 for spleen to 480 000 for liver and to 515 000 for heart apoferritin; moreover, the diffusion coefficients prove that each tissue-specific ferritin consists of a population of hybrid molecules. The intrinsic fluorescence and the near-UV circular dichroism (CD) spectra change as a function of the subunit composition of the three ferritins. The fluorescence emission maximum, which occurs at a very low wavelength (315 nm) in horse spleen apoferritin, is shifted to increasingly higher wavelengths in the liver and heart proteins (320 and 325 nm, respectively), indicating that the tryptophan and tyrosine residues become less rigidly immobilized with an increase in the H-subunit content. The tryptophan residues behave as fully solvated in the monomeric subunits at acidic pH values. In accordance with the fluorescence data, the near-UV CD spectra show that the tryptophan environment is highly asymmetric in spleen apoferritin, progressively less so in liver and heart apoferritins, and completely relaxed in the dissociated subunits. Moreover, they show that the environment of tyrosines and phenylalanines differs markedly in the spleen and heart apoproteins. The ferritins studied appear to have immunogenic sites which are specific for the H and L subunits on the basis of enzyme-linked immunoassay and double-diffusion experiments.

Animals↗
PubMed1982

Catalytic properties of human urinary kallikrein.

Kinetic studies have been carried out with a well-characterized preparation of human urinary (h.u.) kallikrein using chromogenic substrates. Steady-state and pre-steady-state data for h.u. kallikrein catalyzed hydrolysis of N alpha-carbobenzoxy-L-lysine p-nitrophenyl ester (ZLysONp) and of N alpha-carbobenzoxy-L-alanine p-nitrophenyl ester (ZAlaONp) in the presence and absence of ethylamine and acetamidine have been obtained under various conditions and have been analyzed in the framework of the minimum three-step mechanism: (formula see text) The pH dependencies of the kinetic parameters for the hydrolysis of ZLysONp and ZAlaONp in the presence of saturating levels of ethylamine and acetamidine show that at acid pH values (less than or equal to 4) the k3 step (deacylation) is rate limiting in catalysis, whereas for pH values greater than or equal to 6, k2 (acylation) becomes rate limiting. On the other hand, the acylation step is rate limiting in the enzymatic hydrolysis of ZAlaONp over the whole pH range explored. Saturating concentrations of acetamidine increase, more than those of ethylamine, kcat for the hydrolysis of ZAlaONp. The affinity of h.u. kallikrein for acetamidine and ethylamine changes about 5-fold with pH between pH 5 and 3. The pH dependence of the spectral properties of free hu. kallikrein reflects the ionization of a group with a pKa value of 4.45 +/- 0.1. The results point out that, similarly to bovine beta-trypsin, h.u. kallikrein catalysis involves an ionizable group which has a pKa of about 4.5 in the free enzyme and a pKa of about 3.7 in the enzyme bound to cationic substrates or ligands.

Amidines↗
PubMed1982

Catalytic properties of serine proteases. 2. Comparison between human urinary kallikrein and human urokinase, bovine beta-trypsin, bovine thrombin, and bovine alpha-chymotrypsin.

The catalytic properties of several serine proteases acting on cationic substrates (bovine of beta-trypsin, bovine thrombin, human urinary kallikrein, and human urokinase) and noncationic substrates (bovine alpha-chymotrypsin) have been compared in steady-state and pre-steady-state experiments by using ester and anilide synthetic substrates. Arginine and lysine derivatives are equally good substrates for b. beta-trypsin; b. thrombin and h.u. kallikrein prefer substrates containing arginine side chains; h. urokinase prefers substrate containing lysine. The preference of the various enzymes for the guanidinium or ammonium group in reflected by the different promotor effect that acetamidine or ethylamine has on the catalyzed hydrolysis of neutral substrates. Pre-steady-state data, analyzed in the framework of the three-step model, show that for b. beta-trypsin, b. thrombin, h.u. kallikrein, and h. urokinase the acylation step (k2) is rate limiting above pH 6 and the deacylation step (k3) below pH 4 in the hydrolysis of ZLysONp and of ZAlaONp in the presence of acetamidine or ethylamine. In the catalyzed hydrolysis of ZAlaONp, in the absence of acetamidine or ethylamine, the acylation step (k2) is rate limiting all over the pH range from 3 to 8. The change in the rate-limiting step with pH is always absent, for the same substrates, in the b. alpha-chymotrypsin catalysis. The results of kinetic and spectral measurements indicate that b. beta-trypsin, b. thrombin, h.u. kallikrein, and h. urokinase, but not b. alpha-chymotrypsin, contain a similarly located ionizable group with a pKa of 4.50 +/- 0.1, in the free enzyme, the ionization of which affects the binding of cationic substrates and ligands, the spectral properties of the pancreas, and the rate of the acylation step in catalysis.

Animals↗
PubMed1982

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.

Azurin↗
PubMed1982

The effect of pH and D-glycerate 2,3-bisphosphate on the O2 equilibrium of normal and SH(beta 93)-modified human hemoglobin.

The present paper reports data for the effect of pH and D-glycerate 2,3-bisphosphate (D-glycerate-2,3-P2) on the oxygen equilibrium of normal and SH(beta 93)-modified human hemoglobin. At sufficiently high D-glycerate-2,3-P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at all pH values between 6 and 9. Furthermore the difference in the affinity for D-glycerate-2,3-P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK values of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of D-glycerate-2,3-P2 on the hemoglobin molecule. In the hemoglobins modified in position beta 93, the difference in affinity between deoxy and oxy hemoglobin for D-glycerate-2,3-P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for D-glycerate-2,3-P2. The effect of the chemical modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especially pronounced in the presence of phosphates.

2,3-Diphosphoglycerate↗
PubMed1982

Antibacterial activity of matrix-bound ovotransferrin.

Ovotransferrin immobilized by covalent linkage to Sepharose 4B showed a bacteriostatic effect towards Escherichia coli similar to that of free ovotransferrin. The growth of the bacteria, after exposure to the gel-bound ovotransferrin and its removal, depended on the length of exposure. The results suggest that the antibacterial activity of transferrin is not due simply to the removal of iron from the medium.

Anti-Bacterial Agents↗
PubMed1982

Kinetics of reactions within single erythrocytes: studies by microspectrophotometry.

Single cell spectroscopy has been applied to red blood cells along three different lines of research: a) distribution of single hemoglobin components among different erythrocytes, b) chemical reaction with ligands and diffusion in the red blood cell c) dynamics of hemoglobin S aggregation in single red blood cell. The results reported here show, in general, the great possibilities of using single-cell spectroscopy in investigating complex integrated systems.

Anemia, Sickle Cell↗