PubMed Health⌕ Search

Biomedical subjects

F M Veronese

Publications and source records attributed to F M Veronese.

At least 19 recordsLinked to original sources
PubMed1991

Accurate evaluation method of the polymer content in monomethoxy(polyethylene glycol) modified proteins based on amino acid analysis.

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.

Amino Acids↗
PubMed1991

In vitro and in vivo behaviour of narciclasine released from matrices based on poly (2-hydroxyethyl methacrylate).

Narciclasine (1,2,3,7-tetrahydroxy-8,9-methylendioxy-1,2,3,4-tetrahydrophena ntridone) is a natural substance with strong antimitotic effects on cells and potential antitumor activity. Its release form a hydrogel matrix was studied with the purpose of avoiding the concentration spikes of the parenteral administration. The matrix prepared by gamma ray polymerization of a mixture of 2-hydroxyethyl methacrylate (85%) and trimethylolpropane trimethacrylate (15%) was found to release narciclasine for several days, according to a diffusion controlled mechanism. In agreement with its antimitotic effect, narciclasine inhibited the growth rate of healthy mice, when the drug-loaded matrix was introduced subcutaneously. Antitumor effect was observed in an experimental model of Erlich ascitic tumor when low amounts of tumor cells were inoculated. No effect was observed at high concentrations of inoculum or towards solid tumors (Sarcoma 180). This behaviour was related to the rapid clearance of narciclasine from the body which prevented the reaching of sufficient therapeutical concentrations. A pharmacokinetic investigation carried out by an original method of assay demonstrated that narciclasine was accumulated in significant amounts in the kidney only and eliminated in urine with a half time of less than 20 min.

Alkaloids↗
PubMed1979

Drug-protein interaction: plasma protein binding of furocoumarins.

The binding of six furocoumarins (angelicin, psoralen, 8-methoxypsoralen, 5-methoxypsoralen, 8-methylpsoralen, 4,5'8-trimethylpsoralen) to human serum and human serum albumin was studied by equilibrium dialysis using tritium labelled compounds. The results indicated that in serum all furocoumarins are bound mostly by albumin, the extent of binding being related to the structure of the furocoumarins; at any rate, high values of the bound drug, ranging from 84 to 97% were observed. The percentage of binding is strictly related to the water solubility of the compounds. A limited number of binding sites, n = 1-2.4, were detected in the albumin molecule, indicating a high specificity in the binding process. The association constants of the furocoumarins to albumin. Ka, ranged from 1.2 X 10(4) M-1 (8-methoxypsoralen) to 1.9 X 10(5) M-1 (4,5'8-trimethylpsoralen).

Biological Availability↗
PubMed1978

The binding of 8-methoxypsoralen by human serum albumin.

The ability of 8-methoxypsoralen (8-MOP) to bind human serum albumin has been investigated in vitro through equilibrium dialysis and fluorescence quenching. By means of the first technique it was observed that, at concentrations presumably close to those obtainable in vivo following its administration in the photochemotherapy of psoriasis, over 80% of 8-MOP was bound to serum albumin. In human serum albumin fluorescence techniques revealed a preferential site of binding for 8-MOP with a high binding constant (Ka = 0.7 X 10(5) M-1) and precise steric requirements, since small conformational variations of the protein molecule were able to abolish its affinity for furocoumarin.

Dialysis↗
PubMed1977

A fluorimetric study of the role of calcium ions in the stability of thermolysin.

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N'-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn(2+) ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.

Calcium↗
PubMed1977

Fluorescent labelling of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

The thermophilic 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus was inhibited upon specific modification of the -SH group of cysteine residues by 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) at pH 7.0. By using 20-100-fold molar excess of NBD-CL the reaction occurs slowly at pH 7.0 as a first order process. Partial protection from inactivation was observed when the substrate 6-phosphogluconate or the coenzyme NADP was added to the reaction mixture. Complete inactivation was achieved upon modification of 1.9 of the six cysteine residues per mole of enzyme, which corresponds to nearly one residue per enzyme subunit. Circular dichroism measurements suggest that the gross structure of the protein molecule is practically unchanged upon reaction of the enzyme with NBD-Cl. Melting profile experiments revealed a single transition occurring at about 65 degrees C. Analogously, the profile of intensity of the fluorescence emission at 520 nm of the enzyme-bound S-NBD groups versus temperature indicated a midpoint of transition near 65 degrees C. Since this melting temperature corresponds closely to that observed with the native enzyme, these results would indicate that the molecular organizations of the native and modified enzyme are similar and stabilized by similar interactions within the polypeptide chain.

4-Chloro-7-nitrobenzofurazan↗
PubMed1977

Drug-protein interaction: the binding of cephalosporins to albumins.

The binding of some cephalosporin antibiotics, namely cephalothin, cephaloridine and cephalexin, to serum albumins was quantitized using a fluorescence probe technique. The results suggest that these drugs bind to hydrophobic sites on serum albumins. The association constants of the three drugs with bovine serum albumin showed the strongest binding for cephalothin (Ka 1.2 x 10(3) M(1)) and weaker ones for cephaloridine and cephalexin (Ka 0.59 and 0.4 x 10(3) M(-1)). Serum albumin from different species was also investigated, only minor variations in the binding properties being found.

Animals↗
PubMed1976

Isolation and properties of 6-phosphogluconate dehydrogenase from Escherichia coli. Some comparisons with the thermophilic enzyme from Bacillus stearothermophilus.

6-Phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating), EC 1.1.1.44) of Escherichia coli MREp 600 has been isolated with the purpose of carrying out comparative studies with the thermostable enzyme previously isolated from Bacillus stearothermophilus (Veronese, F.M., Boccù, E.,Fontana, A., Benassi,C.A., and Scoffone, E. (1974), Biochim, Biophys. Acta 334, 31). The purified enzyme appeared homogeneous by the criteria of disc gel electrophoresis with and without sodium dodecyl sulfate, ultracentrifugation, and gel filtration. The enzyme has enzymological and physiochemical properties similar to the enzyme isolated from other sources, including B. stearothermophilus. The E. coli enzyme has a mol wt of 100,000 +/- 3000 and is composed of two apparently identical subunits. The amino acid composition of both the mesophilic and thermophilic enzyme has been determined and found to present large similarities. The E. coli enzyme shows a high degree of specificity for nicotinamide adenine dinucleotide (NADP) and it is inhibited by reduced NADP (NADPH). Cysteine residues are involved in the catalytic activity, since on incubation of the enzyme with p-chloromercuribenzoate or 5.5'-dithiobis(2-nitrobenzoic acid) strong inhibition occurs, activity being restored by treatment with excess of beta-mercaptoethanol. The substrate 6-phosphogluconate protects partially the enzyme from inactivation. Both the mesophilic and thermophilic 6-phosphogluconate dehydrogenases are inactivated by Rose Bengal in the presence of light by similar kinetics and protected against photoinactivation by the enzyme substrate. The E. coli enzyme, on the other hand, showed distinct differences in stability against heat and unfolding agents in respect to the B. stearothermophilus enzyme. Heating at 50 degrees C or incubation in 8 M urea results in rapid inactivation. The gross structure of the mesophilic and thermophilic enzyme was very similar as judged by circular dichroic measurements. The far-ultraviolet circular dichroic spectrum had a negative band centered at about 220 nm. In both cases, the fluorescence emission spectrum indicates that the environment of the tryptophan residues is similar, since both enzymes show an emission maximum at 334 nm upon excitation at 295 nm. Circular dichroism measured at various temperatures between 25 and 80 degrees C showed the mesophilic enzyme to be conformationally stable below about 45 degrees C and the thermophilic enzyme below 60 degrees C. The secondary structure of the E. coli enzyme was very sensitive to the denaturing action of urea, since in 8 M urea it rapidly unfolded. Partial renaturation after urea treatment occurred on dilution with buffer or dialysis, as evidenced by spectral properties of the renatured enzyme. The results show that the mesophilic and thermophilic enzymes are very similar and that differences in thermal stability depend on subtle differences in the architectures of the proteins.

Amino Acids↗
PubMed1976

Purification, characteristics and sequence of a peptide containing an essential lysine residue.

Glutamate dehydrogenase (EC 1.4.1.2-4) has been purified and crystallized from the acetone powder of tuna liver. The enzyme has a molecular weight of 333 000 +/- 15 000 as evaluated by sedimentation equilibrium and constists of six identical subunits. Unlike the bovine enzyme the molecular weight does not increase with increasing protein concentration indicating that the tuna enzyme has no tendency to polymerize. The amino acid composition and peptide maps of the tuna and bovine liver enzyme are similar, suggesting considerable homology between the two enzymes. Furthermore, from the tryptic digest a hexadecapeptide containing a lysine residue reactive to pyridoxal 5'-phosphate exhibits the same composition and sequence as the peptide containing the reactive lysine-126 in the sequence of the bovine enzyme. The molecular activity is 25 and 510 mol of substrate per mol enzyme per s, respectively, for the glutamate oxidation and the alpha-ketoglutarate reduction with NAD or NADP as coenzymes. The enzyme is regulated by pyridine nucleotides like other vertebrate enzymes, but it also exhibits some coenzyme specificity, the activity being about fifteen times higher with NAD than with NADP.

Amino Acids↗
PubMed1976

Thermal properties of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli.

The molecular properties of glyceraldehyde 3-phosphate dehydrogenase from E. coli have been evaluated by circular dichroism and fluorescence emission spectroscopy measurements, with the purpose of studying the structural properties which are relevant for a comparison with the enzyme from the obligate thermophile Bacillus stearothermophilus. The enzyme is moderately resistant to heat treatment, being pratically stable when treated for 10 min at 50 degrees C and completely inactivated when heating was performed at 60 degrees C. The secondary structure of the E. coli GPDH appears to be predominatly beta-structure as judged by circular dichroism, showing a negative band centered at about 219 nm. The emission fluorescence of the enzyme shows a maximum at 333 nm upon excitation at 295 nm. In the native E. coli enzyme the tryptophan residues seem to be buried in a hydrophobic region rather than exposed to a polar environment. The structure of the enzyme did not change up to about 50 degrees C, at which temperature thermal inactivation takes place. Upon denaturation the circular dichronic signal at 219 nm gradually decreases, and a red shift of the emission maximum from 333 nm to ca. 345 nm upon heating is indicative that the native structure of the enzyme is unfolded, the tryptophan being exposed to the solvent medium. Since it has been found that the E. coli GPDH closely resembles in many of its properties the B. stearothermophilus enzyme, this bacterial enzyme seems to be useful for comparision with the thermophilic enzyme in studies of its thermostability.

Circular Dichroism↗
PubMed1976

Comparative conformational properties of thermophilic and mesophilic 6-phosphogluconate dehydrogenase.

The structural properties of 6-phosphogluconate dehydrogenase from the mesophilic bacterium E. coli and the thermophilic B. stearothermophilus are compared using circular dichroism and fluorescence emission spectroscopy. The enzymes appear to possess a similar structure which does not change on heating up to the respective temperature of stability of the enzyme. The thermostability of the two 6-phosphogluconate dehydrogenases as determined by activity measurements parallels that determined by CD with the melting profile method, indicating that the loss of biological activity in the enzymes is directly related to the unfolding of the protein molecule. The pattern of unfolding of the proteins by the action of 8 M urea suggests that a core of enhanced conformational stability exists in the B. stearothermophilus enzyme.

Circular Dichroism↗
PubMed1976

[Research on enzymes of Mycoplasma: enolase of Mycoplasma hominis].

A screening of enzymes on cell-free extracts of various species of mycoplasmas revealed the presence of enolase (EC 4.2.1.11) in significative amount in M. pneumoniae and M. fermentans, in lower amounts in M. hominis, A. laidlawii and in trace only in U. urealyticum. The value of activity of the various mycoplasmas could be correlated with their metabolism. From 40 g of cell paste of M. hominis, 2.5 mg of enolase purified over 70 folds, was obtained with successive steps of salt fractionation and column chromatography. Kinetic studies gave the following constants: Km for 2-phospho-D-glicerate, 0.7 x 10(-4)M; optimum of Mg++ concentration, 1 x 10(-3)M; optimum of pH, 7.7 inhibition by fluoride and phosphate, I = 0.77 X 10(-12)M4. Molecular weight estimation indicated for the native enzyme a value of about 100,000 daltons. These data suggest closer similarities with the enolase of microorganisms like E. coli and yeast than with the genus Thermus or B. stearothermophilus.

Mycoplasma↗
PubMed1976

[Drug-protein interactions: physico-chemical study on the interactions between chlorpromazine and glutamate dehydrogenase].

The molecular basis of inhibition of glutamate dehydrogenase by chlorpromazine was studied by circular dichoroism, differential spectroscopy and fluorescence. Chlorpromazine appears to induce conformational changes of the enzyme at the position of aromatic amino acids, and tryptophan residues in particular are involved. On the other hand the secondary structure of the enzyme is not influenced by binding of the drug.

Binding Sites↗