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Biomedical subjects

G Amiconi

Publications and source records attributed to G Amiconi.

At least 91 records · Page 5Linked to original sources
PubMed1985

Kinetics of the reaction of intraerythrocytic haemoglobin by single cell microspectroscopy: effect of shape and osmolarity.

The kinetics of the reaction of CO with intraerythrocytic haemoglobin has been studied in single red blood cells (RBC) using a scanning microspectrophotometer and a photochemical perturbation method. Measurements have been carried out using red blood cells from man and camel (Camelus dromedarius), the latter at different osmotic pressures. Camel RBC, which are smaller and different in shape compared to human RBC, are known to remain intact even at an osmolarity 6-times lower than physiological (280-290 mosm/l), swelling up to twice their normal volume. The results show that the recombination time course is affected by diffusion of CO through a stagnant layer of solvent around the cell membrane, but that it is also influenced by other parameters such as intracellular diffusion of ligand and haemoglobin.

Animals↗
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

Catalytic properties of Ancrod, the thrombin-like proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom.

Kinetics for the hydrolysis of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids catalyzed by Ancrod were determined between pH 5 and 10 (I = 0.1 M) at 21 +/- 0.5 degrees C; the results are consistent with the minimum three-step mechanism: (formula: see text) For all substrates examined, the pH profiles of kcat and/or kcat/Km reflect the ionization of two groups with pKa values ranging between 6.9 and 7.2, and 9.3 and 9.6 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); at variance, values of Km are pH-independent. Moreover, the formation of the E X S complexes may be regarded as a pseudo-equilibrium process, and the acylation step (k + 2) is always rate-limiting in catalysis. Among p-nitrophenyl esters examined, ZArgONp shows the most favourable kinetic parameters and may be the substrate of choice for Ancrod, in that it allows the determination of the enzyme concentration as low as 1 X 10(-9) M (approximately equal to 0.1 Ancrod units/ml), at the optimum pH value (approximately equal to 8). The catalytic behaviour of Ancrod is compared to that of serine proteinases acting on cationic and non-cationic substrates; differences in kinetics, which refer to a lower enzyme:substrate affinity, may be related to a higher rigidity, lower hydrophobicity and/or adverse steric hindrance of the S1 subsite of Ancrod.

Amino Acids↗
PubMed1985

Determination of methionine sulfoxide in proteins: comparison of a gas-chromatographic and electrophoretic method.

Two methods for the determination of methionine in proteins have been used to estimate the extent of methionine sulfoxide obtained upon exposure of proteins to oxidizing agents. Both methods are based on prior treatment with cyanogen bromide, which attacks methionines (but not the sulfoxide derivative) with the resultant formation of methyl thiocyanate and peptides. The amount of methyl thiocyanate is determined quantitatively by gas chromatography, while the number of peptides is ascertained by SDS-polyacrylamide gel electrophoresis. The gas chromatographic estimate of CH3SCN offers an accurate and precise method (down to nanogram values) for the quantitative determination of methionine sulfoxide in proteins. Due to its simplicity and the use of low-cost equipment, the electrophoretic method appears to be a valuable complement to the gas chromatographic method, and the two methods in conjunction provide novel results.

Chemical Phenomena↗
PubMed1984

Different primary specificity of porcine pancreatic beta-kallikrein-B and bovine beta-trypsin. A comparative steady-state and pre-steady-state study.

The values of pre-steady-state and steady-state parameters for the beta-trypsin catalyzed hydrolysis of Z-Arg-ONp and Z-Lys-ONp are superimposable between pH 2.4 and 8. At variance, the kinetic parameters for the beta-kallikrein-B catalyzed hydrolysis of Z-Arg-ONp are more favourable than those observed for Z-Lys-ONp and depend on different pKa values. The different primary specificity and the catalytic behaviour of beta-trypsin and beta-kallikrein-B reflect structural differences at their S1 subsite, especially at level of the 226 residue as well as the 217-220 segment.

Animals↗
PubMed1984

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human urinary kallikrein and to porcine pancreatic beta-kallikreins A and B.

The effect of pH and temperature on the association equilibrium constant (Ka) for bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B has been investigated. Ka values decrease with decreasing pH, reflecting the acid-midpoint and pK shifts, upon BPTI binding, of a three-proton co-operative transition, between pH 3 and 5, and of a single ionizable group, between pH 5 and 9. At pH 8, the values of delta H degree (between 7 degrees C and 42 degrees C) and delta S degree (at 21 degrees C) for BPTI binding to the glandular kallikreins considered were determined. In particular, the delta H degree values have been found to be independent of temperature and the following values have been obtained by van't Hoff plots: +1.8 kcal/mol, +2.3 kcal/mol and +2.4 kcal/mol (1 kcal = 4184 J) for the inhibitor binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B, respectively. Considering the known molecular structures of free porcine pancreatic beta-kallikrein A and BPTI, and of their complex, the stereochemistry of the enzyme : inhibitor contact regions was analysed for the three serine proteinases, in relation to their respective types of behaviour.

Animals↗
PubMed1984

The pH dependence of pre-steady-state and steady-state kinetics for the porcine pancreatic beta-kallikrein-B-catalyzed hydrolysis of N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester.

Pre-steady-state and steady-state kinetics of the porcine pancreatic beta-kallikrein-B (EC 3.4.21.35) catalyzed hydrolysis of ZArgONp have been determined between pH 2.4 and 8. The results are consistent with a minimum three-step mechanism involving an acyl-enzyme intermediate: (see formula). The formation of the E X S complex may be regarded as a pseudoequilibrium process; the minimum values for k+1 are 5.9 X 10(6) M-1 X s-1 (pH 5.5) and 9.4 X 10(5) M-1 X s-1 (pH 2.4) and that for k-1 is 600 s-1. The value of k-1/k+1 (= Ks) changes from 102 microM at pH greater than or equal to 5.5 to 638 microM at pH less than 2.4. The pH dependence of k+2 conforms to two ionizing groups, in the E X S complex, with pKa values of 3.4 +/- 0.1 and 7.05 +/- 0.10. The pH profile of k+2/Ks (= kcat/Km) reflects the ionization of two groups, in the free enzyme, with pKa values of 4.2 +/- 0.1 and 7.05 +/- 0.10. The pH dependence of k+3 implicates two ionizing groups in the deacylation step with pKa values of 4.6 +/- 0.1 and 7.0 +/- 0.1. At acid pH values (pH 2.4-4.4), k+3 is rate-limiting in catalysis, whereas for pH values higher than 4.4, k+2 becomes rate-limiting. The observed neutral and acid ionizations probably reflect the acid-base equilibrium of His-57 and Asp-189 involved in the central site of beta-kallikrein-B. The structural basis for the specificity and catalytic behaviour of this proteinase are discussed and a role for Ser-226 is pinpointed.

Animals↗
PubMed1984

Catalytic and ligand binding properties of bovine trypsinogen and its complex with the effector dipeptide Ile-Val. A comparative study.

Steady-state and pre-steady-state kinetic data for the trypsinogen catalyzed hydrolysis of a series of synthetic substrates (i.e. p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids) have been obtained as a function of pH (3.4-8). Moreover, the effect of ethylamine on the hydrolysis of a neutral substrate and benzamidine binding have been extensively studied. In order to obtain direct information on the transition of trypsinogen to a beta-trypsin-like structure, the role of the effector dipeptide Ile-Val on the catalytic and ligand binding properties of the zymogen has been investigated. Kinetic and thermodynamic data for beta-trypsin and alpha-chymotrypsin are also reported for the purpose of an homogeneous comparison of the various (pro)enzymes. Under all the experimental conditions, kinetic data for (pro)enzyme catalysis are consistent with the minimum three-step mechanism: (formula; see text) involving the acyl intermediate E X P. In the presence of Ile-Val dipeptide, trypsinogen assumes catalytic and ligand binding properties that are reminiscent of activated beta-trypsin. This is at variance with free trypsinogen, which shows a alpha-chymotrypsin-like behavior. The large differences in the results of kinetic and thermodynamic measurements for free trypsinogen, as compared to its binary adduct with Ile-Val, can be ascribed to the substantial differences in the two molecular species, which include the spatial orientation of Asp189.

Animals↗
PubMed1984

Benzamidine as a spectroscopic probe for the primary specificity subsite of trypsin-like serine proteinases. A case for BPTI binding to bovine beta-trypsin.

Formation and dissociation of the benzamidine:beta-trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine:beta-trypsin complex minus the free proteinase) is 1.75 mM-1 cm-1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the beta-trypsin:BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 +/- 0.5 degree C. Under all the experimental conditions the beta-trypsin:BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine:beta-trypsin:BPTI adduct (7). The difference between the kinetic processes of beta-trypsin:BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S1 subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.

Amidines↗
PubMed1984

Possible involvement of amine oxidase inhibition in the sympathetic activation induced by khat (Catha edulis) chewing in humans.

The possibility that phenylalkylamines (cathinone and cathine) of khat, like amphetamines, are amine oxidase inhibitors with clinical significance is evaluated. Results show that khat chewing induces a significant increase in blood pressure, body temperature and urinary catecholamines in khat naive subjects. On the other hand, in habitual consumers a significant enhancement only of blood pressure is observed. Urinary concentrations of vanilmandelic acid show an inhibitory trend in both groups of subjects. This has been related to a decrease in catecholamine deamination. In fact, oxidative deamination of benzylamine catalyzed by beef plasma amine oxidase is non-competitively inhibited in vitro by cathinone (Ki = 0.05 mM) and cathine (Ki = 10 mM) as well as by amphetamine (Ki = 7.9 mM).

Adult↗
PubMed1984

Tolerance develops to sympathetic effects of khat in humans.

Khat chewing has been found to induce, like amphetamines, psychostimulation and increase in sympathetic tone. The possibility that tolerance develops to these effects has been studied by comparing the influence of the chewing of a standard dose of khat on some physiological parameters in chronic consumers and naive subjects. Systolic blood pressure appears to rise in both groups, but the increase in diastolic pressure was more marked in naive subjects and inversely related to the reduction of pulse rate. The increases of respiratory rate and body temperature were also higher in naive subjects. In both groups, all these effects disappeared after 18 h. These results are consistent with an amphetamine-like mechanism of action and suggest that tolerance develops to the sympathetic effects of khat.

Adult↗
PubMed1984

Aromatic tetra-amidines: synthesis of halo-derivatives and their antiproteolytic activity.

Mono-halo derivatives of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)p ropane (TAPP-H) have been synthesized and their inhibitory effect on the bovine trypsin, bovine thrombin and porcine pancreatic kallikrein catalyzed hydrolysis of p-nitroanilides of amino acids was investigated, at pH 8.1 and 37 degrees, in parallel with that of TAPP-H and benzamidine. The addition of a halogen (Cl, Br or I) at position 2 of each benzamidine moiety of TAPP-H is accompanied by an increase of the inhibitory effect. This is especially evident in the case of porcine pancreatic kallikrein inhibition by TAPP-Cl. The structural basis of the different inhibitory effect of TAPP-H, TAPP-halo derivatives and benzamidine on the catalyzed hydrolysis of the proteinases examined are discussed and the role of Asp-189 in bovine trypsin, Asp-189 and Glu-149 or Asp-192 in bovine thrombin and Asp-189 and Asp-A148 or Glu-150 in porcine pancreatic kallikrein is focused.

Amidines↗
PubMed1982

Structural and functional changes in man accompanying the weightlessness in "Skylab" flights: a mathematical approach.

The present paper reports a kinetic analysis of changes of some physiological parameters, obtained from international literature, after changes in gravitational environment. The overall phenomenology of the adaptation to weightlessness is characterized by a rapid process followed by a slow one. The two processes show half time values differing by about five times. Also in the case of readaptation to gravity, after recovery on the Earth, two well resolved processes, showing different half time values, are observed. It is of interest to notice that the rate of response to weightlessness is lower than that to gravity. Of course, the half time values observed depend on the different physiological parameters considered. In any case, the experimental data suggest a general trend of many adaptive changes, that may all be described by a simple mathematical model.

Adaptation, Physiological↗
PubMed1981

Binding of organic ions by proteins. 2. Effects of 1-benzyl-3-indazoleoxyacetate at low concentration on the oxygen affinity of human hemoglobin.

The oxygen equilibrium of human hemoglobin has been studied in the presence of 1-benzyl-3-indazoleoxyacetate (BZ). The results show that: (a) The overall oxygen affinity of hemoglobin is a function of BZ concentration, but the cooperative character of the equilibrium curve appears insensitive to the drug up to the maximal concentration studied (5 x 10(-2) M); (b) The functional expression of the interaction between hemoglobin and BZ is not affected by the presence of protons, i.e., the change in oxygen affinity determined by BZ is the same at any pH value studied; (c) The aromatic region of BZ molecules is of primary importance for the functional change of hemoglobin; (d) The difference in moles of BZ bound per mole of tetrameric unliganded and oxygenated hemoglobin corresponds to 2; these functionally relevant binding sites on the protein are probably located at alpha 1, beta 1 and alpha 2 beta 2 interfaces; (e) Thermodynamically, entropy effects dominate the reaction between hemoglobin and BZ.

Dose-Response Relationship, Drug↗
PubMed1981

Evidence for multiple differing cation binding sites on hemoglobin A and S.

Some divalent cations (at 10(-3)M concentration) have been shown to act as oxygen-linked ligands. The effect of Ca++ (only on hemoglobin S) and Zn++ (on normal hemoglobin and hemoglobin S) can be traced to a preferential binding of these cations to the oxy- conformation of the proteins; the reverse is true for Ni++, since its presence reduces the oxygen affinity of both hemoglobins. The results suggest: i) the existence of at least two different cation binding sites on hemoglobin; ii) the replacement of glutamic acid by valine in hemoglobins S introduces sufficient structural modification to form a new cation binding site.

Binding Sites↗