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Biomedical subjects

E Menegatti

Publications and source records attributed to E Menegatti.

At least 127 records · Page 7Linked to original sources
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

pH and temperature effects on the molecular conformation of the porcine pancreatic secretory trypsin inhibitor as detected by hydrogen-1 nuclear magnetic resonance.

1H NMR spectra of the porcine pancreatic secretory trypsin inhibitor (PSTI) have been recorded vs. pH and temperature. Of the two tyrosines, one titrates with a pK of 11.25, while the resonances from the other are pH insensitive in the investigated range 4.8 less than or equal to pH less than or equal to 12. This is consistent with PSTI having one Tyr solvent exposed (Tyr-20) and the other buried (Tyr-31). The resonances from the lysyl epsilon-CH2 protons titrate with a pK of 10.95. The titration is accompanied by a pronounced line broadening, which starts near pH 8.5. Between pH 11.5 and pH 12 the epsilon-CH2 resonances recover their low pH line width. Titration curves for the lysines and Tyr-20 reflect single proton ionization equilibria, suggesting that these residues do not interact among themselves. On the basis of double resonance experiments, combined with analysis of chemical shifts, spin-spin couplings, and line widths, all methyl resonances are identified and followed as functions of pH and temperature. The gamma-CH3 doublet from the N-terminal Thr-1 is assigned by comparison between spectra of forms I and II of the inhibitor, the latter lacking the first four residues of form I. The beta-CH3 resonance from Ala-7 is also assigned. Proton resonance parameters of methyl groups are shown to afford useful NMR probes for the characterization of local nonbonded interactions, microenvironments, and mobilities.

Alanine↗
PubMed1981

[Action of the basic trypsin inhibitor on lymphocyte blastic transformation in vitro].

The effect of Basic-trypsin-inhibitor (BPTI) on the in vitro PHA-induced blastic transformation of lymphocytes is reported. Cultures of lymphocytes from healthy subjects were added with PHA, BPTI and PHA + BPTI; simple control-cultures were also performed. The blastic transformation was evaluated by morphologic method after 72 h incubation at 37 degrees C. The results are compared with Wilcoxon's test; it was observed that BPTI reduces the PHA-induced blastic transformation, but it has no effect on cultures without PHA. The hypothesis that BPTI inhibits only PHA-stimulated lymphocytes is suggested.

Humans↗
PubMed1981

Steady-state and pre-steady-state kinetics of the trypsin-catalysed hydrolysis of alpha-CBZ-L-lysine-p-nitrophenyl ester.

The catalytic properties of bovine tyrpsin (EC 3.4.21.4) have been investigated using a synthetic chromogenic substrate: alpha-CBZ-L-lysine-p-nitrophenyl ester (ZLNPE). The use of ZLNPE allows the determination of trypsin down to a concentration of 2 . 10(-9) M. Steady-state and pre-steady-state data have been in the framework of the minimum three-step mechanism: (Formula: see text). The pH-dependence of the kinetic parameters shows that at acid pH values (congruent to 2.6) the k+3 step is rate limiting in catalysis, whereas for pH values higher than 4.8 k+2 becomes rate limiting. This change in rate-limiting step with pH illustrates the danger in the assumption that kcat vs. pH profiles for protease action on substrates with good leaving groups are equivalent to k+3 vs. pH profiles.

Animals↗
PubMed1979

1H nuclear-magnetic-resonance studies of the porcine-pancreatic secretory trypsin inhibitor at 270 MHz.

The pancreatic secretory trypsin inhibitor from porcine pancreas has been investigated by high-resolution 1H nuclear magnetic resonance (NMR) at 270 MHz. The presence of a number of slowly exchanging labile protons indicates that the protein is highly globular. Of the two tyrosyl rings, one is free-rotating and solvent-exposed while the other one is hindered in its mobility and buried in the interior of the protein. A lineshape analysis of the temperature dependence of aromatic resonances gave the dynamic parameters for activation of ring mobility. The inhibitor exhibits at least three well-resolved high-field ring-current-shifted methyl resonances. Form II of the inhibitor, that lacks the first four residues, has been compared with the intact form I. No detectable differences were found between the spectra of I and II, which indicates that the presence of the N-terminal tetrapeptide does not appreciably affect the overall conformation of the protein.

Animals↗
PubMed1977

Arginine modification in Kunitz bovine trypsin inhibitor through 1, 2-cyclohexanedione.

Arginine residues (5.5 out of 6) of the trypsin-kallikrein inhibitor from bovine organs (Kunitz inhibitor) were selectively modified by reaction with 1, 2-cyclohexanedione in sodium borate buffer, pH 9.0. The modified inhibitor is still highly active in inhibiting trypsin and chymotrypsin at 1:1 inhibitor: enzyme molar ratio and full inhibition was achieved at slightly higher molar ratio. The extent of correct refolding, upon reoxidation, of the reduced, arginine-modified inhibitor is diminished and regeneration of two arginines occurred under the reduction conditions. The stability constants and the standard-free energies of binding of the complexes between trypsin, or chymotrypsin, and the native, the arginine-modified and the reduced and reoxidized arginine-modified inhibitor have been determined from inhibitory assays.

Amino Acids↗
PubMed1976

On the reaction of acetamidomethanol with native and reduced bovine pancreatic trypsin inhibtor (Kunitz inhibitor).

The stability of native and reduced bovine pancreatic trypsin inhibitor (Kunitz inhibitor) in anhydrous hydrogen fluoride and their reaction with acetamidomethanol, in the same solvent, have been investigated. The bovine Kunitz inhibitor appears to be stable in liquid hydrogen fluoride but the reduced molecule loses about 50% of its ability to regain inhibitory power, upon air oxidation, by exposure to this solvent. Tyrosine residues appear to be affected by acetamidomethylation of the native protein to give a modified inhibitor which is still highly active in inhibiting trypsin. The extent of correct refolding, upon reoxidation, of the reduced tyrosine modified-inhibitor is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in the presence of excess anisole. The stability constants and the standard free energies of binding of the complexes between trypsin and the native and the tyrosine modified-inhibitor have been determined.

Acetamides↗
PubMed1976

Studies on trypsin inhibitors. Part V. Synthesis of the protected octapeptide (sequence 36-43) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the partially protected octapeptide tert-butyloxycarbonyl-gamma-tert-butylglutamylasparaginyl-N-trifluoroacetyllysyl-N-trifluoracetyllysylarginyl-Ngamma-4,4'-dimethoxybenzhydryglutaminylthreonylproline corresponding to positions 36-43 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The tetrapeptide free base arginyl-Ngamma-4,4'-dimethoxybenzhydrylglutaminylthreonylproline was acylated, by the azide proceedure, with the tripeptide benzyloxycarbonyl-asparaginyl-N-trifluoroacetyllsyl-N-trifluoroacetyllysine hydrazide. The resulting protected heptapeptide was partially deblocked by catalytic hydrogenation and reacted with alpha-1-succinimidyl-gamma-tert-butyl tert-butyloxycarbonylglutamate. The stereochemical homogeneity of the ensuing octapeptide was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.

Amino Acid Sequence↗