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Biomedical subjects

M Guarneri

Publications and source records attributed to M Guarneri.

At least 73 records · Page 4Linked to original sources
PubMed1987

The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester.

Pre-steady-state and steady-state kinetics of the papain (EC 3.4.22.2)-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester (ZGlyONp) have been determined between pH 3.0 and 9.5 (I = 0.1 M) at 21 +/- 0.5 degrees C. The results are consistent with the minimum three-step mechanism involving the acyl X enzyme intermediate E X P: (Formula: see text). The formation of the E X S complex may be regarded as a rapid pseudoequilibrium process; the minimum values for k+1 are 8.0 microM-1 s-1 (pH less than or equal to 3.5) and 0.40 microM-1 s-1 (pH greater than 6.0), and that for k-1 is 600 s-1 (pH independent). The pH profile of k+2/Ks (= kcat/Km; Ks = k-1/k+1) reflects the ionization of two groups with pK' values of 4.5 +/- 0.1 and 8.80 +/- 0.15 in the free enzyme. The pH dependence of k+2 and k+3 (measured only at pH values below neutrality) implicates one ionizing group in the acylation and deacylation step with pK'' values of 5.80 +/- 0.15 and 3.10 +/- 0.15, respectively. As expected from the pH dependences of k+2/Ks (= kcat/Km) and k+2, the value of Ks changes with pH from 7.5 X 10(1) microM (pH less than or equal to 3.5) to 1.5 X 10(3) microM (pH greater than 6.0). Values of k-2 and k-3 are close to zero over the whole pH range explored (3.0 to 9.5). The pH dependence of kinetic parameters indicates that at acid pH values (less than or equal to 3.5), the k+2 step is rate limiting in catalysis, whereas for pH values higher than 3.5, k+3 becomes rate limiting. The observed ionizations probably reflect the acid-base equilibria of residues involved in the catalytic diad of papain, His159-Cys25. Comparison with catalytic properties of ficins and bromelains suggests that the results reported here may be of general significance for cysteine proteinase catalyzed reactions.

Catalysis↗
PubMed1987

Synthesis and antifungal activity of 4-thiazol-2-yl-5-aminopyrazoles and 4-aminopyrazolo [3,4-c] isothiazoles.

Starting from 4-thiocarbamoyl-5-aminopyrazoles (I), the AA could prepare two series of products: 4-thiazol-2-yl-5-aminopyrazoles (II) and 4-aminopyrazolo [3,4-c] isothiazoles (III). The screening for in vitro antifungal activity evidenced that compounds (III) are effective in controlling most examined fungi; in particular 1-phenyl-3-methyl-4-aminopyrazolo [3,4-c] isothiazole (III a) at the concentration of 20 p.p.m. completely inhibited the growth of Pythium ultimum and Corticium solani, responsible for the collar rot of the beet.

Antifungal Agents↗
PubMed1987

Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane: thermodynamic and molecular modeling study.

The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of beta-trypsin (EC 3.4.21.4), alpha-thrombin (EC 3.4.21.5), factor Xa (EC 3.4.21.6), Lys77-plasmin (EC 3.4.21.7) and beta-kallikrein-B (EC 3.4.21.35) was investigated (between pH 2 and 8, I = 0.1 M; T = 37 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, TAPP and benzamidine show the same values of the dissociation inhibition constant (Ki) for beta-trypsin; at variance with the affinity of TAPP for alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B which is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, values of Ki for TAPP binding to beta-trypsin as well as for benzamidine association to all the enzymes investigated decreased thus reflecting the pK-shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for TAPP binding to alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B may be described as depending on the pK-shift, upon inhibitor association, of two equivalent proton-binding amino acid residues. Considering the X-ray three-dimensional structures and the computer-generated molecular models of serine proteinases: TAPP and :benzamidine adducts, the observed binding behaviour of TAPP and benzamidine to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).

Amidines↗
PubMed1987

[Ethyl esters of N-amidinobenzoyl aminoacids: inhibitory effect on thrombin, blood coagulation and platelet aggregation].

In a previous paper we described the synthesis and the antiproteolytic activity of N-(3- and N-(4-amidinobenzoyl)-L-amino acids. As an extension of these studies we examined the possible inhibitory effect of these compounds on thrombin, blood coagulation and platelet aggregation. Values of Ki for the binding to thrombin are independent of the nature of the amino acids side chain and superimposable with those obtained for the formation of benzamidine-thrombin adduct. These data suggest slight effect on blood coagulation. The benzamidine derivatives are more active on platelets.

Amidines↗
PubMed1986

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys77-plasmin.

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI Kunitz inhibitor) to human Lys77-plasmin has been investigated. Ka values decrease with decreasing pH, reflecting the acid-pK and -midpoint shifts, upon BPTI binding, of a single ionizable group, between pH 5 and 9, and of a three-proton transition, between pH 3 and 5. At pH 8.0, values of thermodynamic parameters for BPTI binding to human Lys77-plasmin are: Ka = 1.2 X 10(9) M-1, delta G degree = -12.2 kcal/mol, and delta S degree = +49 entropy units (at 21 degrees C); and delta H degree = +2.3 kcal/mol (temperature independent between 5 degrees C and 45 degrees C; 1 kcal = 4184 J). BPTI binding properties of human Lys77-plasmin have been analysed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates. Considering the known molecular structures of homologous serine (pro)enzymes, or Kunitz and Kazal-type inhibitors and of their complexes, the observed binding behaviour of BPTI to human Lys77-plasmin was related to the inferred stereochemistry of the enzyme-inhibitor contact region.

Animals↗
PubMed1986

Catalytic properties of bovine alpha-thrombin: a comparative steady-state and pre-steady-state study.

Values of steady-state and pre-steady-state parameters for the bovine alpha-thrombin-catalyzed hydrolysis of ZArgONp and ZLysONp have been determined between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degree C. Kinetic properties of bovine alpha-thrombin have been analyzed in parallel with those of porcine pancreatic beta-kallikrein-B and bovine beta-trypsin, all acting on cationic substrates. The different primary specificity and catalytic behaviour of these three serine proteinases reflect subtle structural differences at their S1 subsite, especially at residue positions 190, 221 and 226 as well as in the 217-219 segment.

Animals↗
PubMed1986

Primary specificity of ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom.

Values of steady-state and pre-steady-state parameters for the hydrolysis of ZArgONp and ZLysONp catalysed by ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom, have been determined, between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C, and analysed in parallel with those of bovine alpha-thrombin and porcine pancreatic beta-kallikrein-B. In addition to the well-known coagulating behaviour, ancrod also shows catalytic properties, in the hydrolysis of ZArgONp and ZLysONp, reminiscent of those of porcine pancreatic beta-kallikrein-B.

Ancrod↗
PubMed1986

Gabexate mesylate inhibition of serine proteases: thermodynamic and computer-graphics analysis.

The inhibitory effect of gabexate mesylate, which is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation, and as a regional anticoagulant agent for hemodialysis, has been measured on bovine factor Xa, bovine alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, human urokinase, porcine pancreatic beta-kallikrein-B, and bovine beta-trypsin catalyzed hydrolysis of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-arginine and N-alpha-carbobenzoxy-L-lysine. On the basis of enzyme:gabexate mesylate affinities, the serine proteases can be arranged as follows: human urinary kallikrein approximately porcine pancreatic beta-kallikrein-B much less than bovine beta-trypsin approximately bovine factor Xa approximately human Lys77-plasmin approximately human urokinase approximately bovine alpha-thrombin. The mode of binding of gabexate mesylate to the serine proteases conforms to the active-reactive site geometries observed in their complexes with natural and synthetic inhibitors. Differences in gabexate mesylate affinities for these proteases reflect structural differences at their primary specificity subsite, which have been investigated by comparative analysis of amino acid sequences and by computer-graphics techniques.

Animals↗
PubMed1986

Aromatic tetra-amidines: antiproteolytic and antiesterolytic activities towards serine proteinases involved in blood coagulation and clot lysis.

The inhibitory effect of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H), TAPP-halo derivatives (with Cl, Br or I) and benzamidine on the Ancrod, bovine Factor Xa, and human plasmin catalyzed hydrolysis of esters and anilides of amino acids was investigated, at pH 8.1 and 37 degrees, and compared with that shown from these compounds on bovine thrombin and porcine pancreatic kallikrein catalysis. The inhibitory effect of TAPP-H and TAPP-halo derivatives on the proteinases considered, involved, to different extents, in blood coagulation and clot lysis, is higher by at least 10-fold, than that of benzamidine, which binds at the primary specificity subsite (S1) of serine endopeptidases and is commonly taken as a molecular inhibitor model. The high inhibitory effect of aromatic tetra-amidines has been interpreted taking into account an additional productive binding for a second benzamidine or halo-benzamidine moiety to the enzyme surface. Moreover, the data reported here allowed us to clarify the inhibition mechanism (in vitro) of TAPP-H on blood coagulation, induced by the "cancer coagulation factor" produced by the Walker carcinoma in Wistar rats and on the fibrinogen-to-fibrin conversion, and to identify some serine proteinases which act as targets for aromatic tetra-amidines.

Amidines↗
PubMed1986

Guanidinophenyl derivatives of pyrazole: synthesis and inhibitory effect on serine proteinases, blood coagulation and platelet aggregation.

Guanidinophenyl derivatives of pyrazole have been synthesized. Their inhibitory effects on (i) bovine trypsin, bovine thrombin, porcine pancreatic kallikrein catalyzed hydrolysis of p-nitro anilide of N alpha -benzoyl-arginine and (ii) blood coagulation and platelet aggregation, were investigated. The kinetic behaviour of all compounds conformed to that of a reversible competitive inhibition pattern, and they were also found to act in vitro as inhibitors of platelet aggregation induced by ADP.

Animals↗
PubMed1985

Trypsin activation. Effect of the Ile-Val dipeptide concentration on Kazal inhibitor binding to bovine trypsinogen.

The effect of Ile-Val concentration (up to 2.0 M) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) to trypsinogen has been investigated at pH 5.5 between 7 degrees C and 42 degrees C. Thermodynamic parameters for Kazal inhibitor binding to the Ile-Val:zymogen adduct are more favorable than those observed for inhibitor association to the free proenzyme, but less so than those reported for beta-trypsin:Kazal inhibitor adduct formation (even under saturating dipeptide concentrations), suggesting that the effector dipeptide does not induce a complete rigidification of the proenzyme's activation domain. Considering the dependence of the association equilibrium constant for Kazal inhibitor binding to trypsinogen from Ile-Val concentration, thermodynamic parameters for the effector dipeptide binding to the free proenzyme and to its binary complex with Kazal inhibitor have been obtained. Differences in affinity for Ile-Val binding to the free zymogen and its binary complexes with inhibitors and substrates are indicative of the presence of different activation levels of the proenzyme, none of them exactly coincident with that of beta-trypsin. Such different discrete states should correspond to those involved in the zymogen-to-active-enzyme transition which should not be considered as an all-or-nothing process, but as a multistep event.

Animals↗
PubMed1985

Catalytic properties of human Lys77-plasmin. A comparative steady-state and pre-steady-state study.

Values of kinetic parameters for the hydrolysis of esters and p-nitroanilides of L-lysine and L-arginine catalyzed by the Lys77 form of human plasmin (EC 3.4.21.7) have been determined between pH 5.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C. Over the whole pH range explored, Lys77-plasmin catalysis conforms to simple Michaelis-Menten kinetics, and steady-state and pre-steady-state data may be consistently fitted to the minimum three-step mechanism: E + S in equilibrium (k+1/k-1)E X S----(k+2)E X P + P1----(k+3)E + P2 In spite of the higher specificity of lysyl derivatives for Lys77-plasmin rather than the arginyl ones, kinetic parameters also depend on the nature of the N-alpha substituent and/or of the alcoholic or p-nitroanilidic moiety of the substrate. Among the esters and anilides considered, ZLysONp shows the most favourable kinetic parameters and may be the substrate of choice of Lys77-plasmin, in that it allows the determination of the enzyme concentration as low as 2 X 10(-9) M (about 1 X 10(-3) CU/ml), at the optimum pH value (approx. 8). Between pH 5.5 and 8, the pH profiles of kcat and kcat/Km for the Lys77-plasmin-catalyzed hydrolysis of ZLysONp and ZArgONp reflect the ionization of a single group (probably His-602 involved in the active site) with pKa values ranging between 6.4 and 6.6; at variance, values of Km are pH-independent.

Catalysis↗
PubMed1985

Activating effect of the Ile-Val dipeptide on the catalytic properties of bovine trypsinogen.

The dependence of pre-steady-state and steady-state kinetics for the trypsinogen-catalyzed hydrolysis of ZArgONp on the concentration (up to 2.0 M) of the Ile-Val effector dipeptide has been investigated at pH 8.0 and 21 +/- 0.5 degrees C. Kinetic parameters for the hydrolysis of ZArgONp catalyzed by the Ile-Val:zymogen adduct are more favorable than those observed for the free proenzyme but lower than those reported for beta-trypsin; these data indicate that the effector dipeptide induces only a partial activation of the zymogen even under saturating Ile-Val concentrations. From the dependence of kinetic parameters of proenzyme catalysis on the effector dipeptide concentration, values of the equilibrium constants for binding of Ile-Val to the free trypsinogen, to the reversible zymogen-ZArgONp complex and to the proenzyme-ZArg acyl intermediate have been obtained. Thermodynamics of binding of Ile-Val to trypsinogen, in the presence and absence of substrates and inhibitors, are indicative of the presence of different activation levels of the proenzyme, none of which is superimposable on that of beta-trypsin. On this basis, it is suggested that some of these different states could correspond to those involved in the zymogen-to-active-enzyme transition, which should be considered as a multistep process, rather than an all-or-nothing event.

Animals↗