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Biomedical subjects

M Guarneri

Publications and source records attributed to M Guarneri.

At least 91 records · Page 5Linked to original sources
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

Synthesis and anti-inflammatory and analgesic activities of 3-methyl-N-phenyl-1H-pyrazol-5-ylcarboxamides.

The anti-inflammatory and analgesic activities of a series of 3-methyl-N-phenyl-1H-pyrazol-5-ylcarboxamides were investigated and compared with flufenamic acid. The compounds were synthesized by condensation of diketopiperazines 2 with the appropriate aniline. The pharmacological tests showed that some compounds have good anti-inflammatory activity in rat paw edema induced by carrageenin and low toxicity.

Animals↗
PubMed1985

Synthesis and interaction of 5-(substituted-phenyl)-3-methyl-6,7-dihydropyrazolo[4,3-e] [1,4]diazepin-8(7H)-ones with benzodiazepine receptors in rat cerebral cortex.

On the basis of the anxiolytic property of ripazepam, 1-ethyl-4,6-dihydro-3- methyl-8-phenylpyrazolo[4,3-e][1,4]diazepin-5(1H)-one (1), a series of isomeric 5-(phenyl-substituted)pyrazolo[4,3-e][1,4] diazepin-8-ones 3a-f were prepared and tested for their ability to bind to the benzodiazepine receptor. All compounds 3a-f display affinities for the benzodiazepine receptor in the microM range of concentration; in particular 5-phenyl-3-methyl-6,7-dihydropyrazolo[4,3-e][1,4] diazepin-8(7H)-one (3a) is 2 orders of magnitude less potent in inhibiting [3H]flunitrazepam binding than diazepam and displays an affinity for the benzodiazepine receptor practically comparable to that of its structural isomer, ripazepam, and to that of chloriazepoxide.

Animals↗
PubMed1985

Ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids: synthesis and antiproteolytic activity towards bovine trypsin and porcine pancreatic kallikrein.

Ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids (namely, glycine, alanine, valine, leucine and phenylalanine) were synthesized and their inhibitory effect on the bovine trypsin and porcine pancreatic kallikrein catalyzed hydrolysis of p-nitroanilides of amino acids was investigated, at pH 8.1 and 37 degrees, in parallel with the effect of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids and benzamidine. For both proteinases, the inhibitory effect of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids is independent of the aminoacidic side chain, is closely similar to that of benzamidine (which binds at the S1 subsite of the proteinases examined and is commonly taken as a molecular inhibitor-model), and is higher by at least 10-fold than that of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids (depending on the aminoacidic residue). On structural grounds, the peculiar inhibitory behaviour of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids has been related to the interaction of the positively charged substituent at the N-position with the Asp189 residue present in the primary specificity subsite (S1) of bovine trypsin and porcine pancreatic kallikrein. The consistently lower affinity for porcine pancreatic kallikrein of all the inhibitors considered, as compared to bovine trypsin, may be related to the marked structural differences of the primary specificity subsite of these two serine proteinases.

Amidines↗
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

Placental transfer of pinazepam and its metabolite N-desmethyldiazepam in women at term.

Placental transfer of pinazepam and its metabolite N-desmethyldiazepam was investigated in 25 pregnant women at term. Pinazepam was administered orally as a single (10 mg) dose to 13 women, or in multiple doses of 5 mg daily to 12 women. The dose-delivery interval ranged between 1 and 26 h for the single dose, and the period between the last of the multiple doses and delivery was 1.4 to 24 h. Pinazepam and N-desmethyldiazepam were measured in plasma obtained from the umbilical vein and from the mother, at delivery. Pinazepam was only detectable in plasma after the 10 mg dose. The drug did not reach an apparent equilibrium between fetal and maternal plasma. The average (+/- SEM) cord/maternal ratio of plasma pinazepam concentrations was 0.64 +/- 0.07. N-desmethyldiazepam was detectable on each occasion. Its concentration in the plasma from the cord vein became higher than that in the maternal specimens 1-2 h after administration of the parent drug. Little N-desmethyldiazepam was excreted in breast milk.

Adult↗
PubMed1984

Pyrazolo[4,3-d]pyrimidine nucleosides. Synthesis and antiviral activity of 1-beta-D-ribofuranosyl-3-methyl-6-substituted-7H-pyrazolo[4,3-d]pyr imi din-7-ones.

N1 analogues of formycin B, with substituents at the 3 and 6 positions of the pyrazolo[4,3-d]pyrimidine moiety were synthesized by the direct SnCl4-catalyzed ribosylation method. The site of the glycosidic linkage and the anomeric configurations were established on the basis of X-ray crystallography, as well as 1H and 13C nuclear magnetic resonance spectroscopy. Preliminary results of the antiviral testing of these derivatives in vitro are described.

Animals↗
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↗
PubMed1983

Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

Acridines↗
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↗