Biomedical subjects
Publications and source records attributed to M Guarneri.
The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.
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Three series of pyrazole derivatives (III a-g), (IV a-g) and (V a-g) were synthesized and tested for analgesic, antipyretic and antiinflammatory activity. Many of tested compounds showed interesting analgesic and antipyretic activity, whereas no compounds exhibited any antiinflammatory activity.
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Ornithine transcarbamylase (OCT) is one of the urea-cycle enzymes in mitochondria which is involved in metabolic disorders in man. Reye's syndrome, characterized by encephalopathy and fatty infiltration of the liver, is one of such diseases. During this syndrome a reduction of OCT specific activity was observed, probably due to increased proteolytic degradation or to alteration in the enzyme conformation. It has recently been found that OCT administration to patients with liver disorders, contributes to illness resolution. Since the therapeutic properties of the enzymes are usually related to their structure, this paper reports some findings obtained by spectroscopic measurements on OCT structure and conformation stability in solution.
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The tertiary structure of the isoinhibitor A (Kazal) isolated from bovine pancreatic tissue has been characterized by 1H-NMR studies in 2H2O solution. A number of slowly exchanging backbone amides are observed. The dynamics of the aromatic side chains and their interaction with methyl groups from aliphatic residues are essentially identical in the bovine and in the homologous inhibitor from porcine pancreas. Both inhibitors contain two tyrosines in positions 20 and 31 as the only aromatic residues. The previous assignment of the aromatic resonances in the porcine inhibitor is valid for the bovine homolog, as deduced from selective nuclear Overhauser enhancement (NOE) experiments. The tyrosine which inhibits an NMR spectral pattern characteristic of rapid ring motion does not show appreciable NOE to backbone or sidechain protons, in agreement with the solvent exposure predicted for Tyr-20 in the porcine inhibitor. In contrast, the immobilized Tyr-31 exhibits a NOE pattern that indicates close interaction with a number of backbone amide protons and aliphatic side chain groups, confirming that the aromatic ring is buried. At 90 degrees C, pH 5, the protein is still substantially folded, as manifested by both backbone and side chain resonances. The results are compared with previous optical rotatory dispersion findings. A number of side chain resonances were assigned to specific amino acid residues in the sequence.
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.
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.
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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.
The synthesis of pyrazolyl-amides of aminoacids and peptides is described. The chemicals were tested for antifungal activity against wheat powdery mildew (Erysiphe graminis DC.), cucumber powdery mildew (Erysiphe cichoracearum DC.), wheat brown rust (Puccinia recondita Rob. ex Desm. f. sp. tritici Erikss et Henn.), celery leaf spot (Septoria Apii Briosi ed Cav. Chest.) and collar rot (Rhizoctonia solani Kuhn). Some of these compounds showed antifungal activity.
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.
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New nitrogen analogues of prostaglandins (11, 11a, 12, and 12a) have been synthesized starting from a 4,5-disubstituted 2-pyrrolidinone nucleus (5 and 5a) containing one side chain and a suitable functionality for elaborating the second one. These analogues had no better activity than natural prostaglandins in vitro [guinea pig ileum and trachea, rat stomach fundus strip, uterus and portal vein, ADP-induced guinea pig platelet-rich plasma (PRP) aggregation]. They similarly lacked any interesting activity in vivo [anesthetized rat blood pressure, stress, and acetylsalycilic acid (ASA) induced gastric lesions in rat].
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The synthesis of 4-nitroso-5-amminopyrazoles and of 4-nitroso-5-pyrazolylurethans and -ureas is described. The chemicals were tested for antifungal activity against Erysiphe graminis, Erysiphe cichoracearum, Puccinia recondita, Septoria apii and Rhizoctonia solani. A number of the described compounds showed some antifungal activity.
4-Oximino-pyrazol-5-ones 2a, b and their esters with N-protected amino acids 3a, b have been studied for carboxyl activation in peptide synthesis. 2a, b and 3a, b appear to be diastereoisomeric mixtures whose configurations have been assigned on the basis of spectroscopic and X-ray data. Some peptides obtained using these pyrazolone derivatives are reported: no racemization was noted by the Weigand test.