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Biomedical subjects

F M Veronese

Publications and source records attributed to F M Veronese.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1975

Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme.

When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced. The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30%. The enzyme proved to be heat stable and relatively resistant to protein denaturants. The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination. The activity is affected by adenine nucleotides. The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin.

Amino Acids↗
PubMed1975

Denaturation of thermophilic and mesophilic 6-phosphogluconate dehydrogenase by 8M urea.

The denaturation reaction of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase (decarboxylating EC 1.1.1.44) from B. Stearothermophilus and E. coli in 8 M urea has been compared. The rates of denaturation were evaluated from the fluorescence quenching that occurs at 334 nm. The thermophilic enzyme has been found about ten times more resistant to the action of the denaturating agent, and showed a biphasic denaturation process, whereas the E. coli enzyme followed a single first-order kinetics.

Escherichia coli↗
PubMed1975

Selective separation of tryptophan derivatives using sulfenyl halides.

Upon reaction of tryptophan containing proteins with 2-nitrophenylsulfenyl chloride (NPS-Cl) the nitroaryl chromphore (lambda max 365 nm, epsilon 4000) is covalently linked at the 2-position of the indole ring of tryptophan. From the mixture of peptides obtained by enzymatic or chemical fragmentation of the labelled protein, the yellow tryptophan peptides are much more easily separated by column as well as paper chromatographic techniques, since their easier detection by spectrophotometry. Tryptophan peptides have been also isolated by diagonal electrophoresis, upon introduction of a carboxylate anion in the peptide by reaction with the carboxy-substituted reagent 2-nitro-4-carboxyphenylsulfenyl chloride. Sulfenl halides have been also used for the selective chemical coupling of tryptophan peptides to a solid support, thus facilitating their separation. N-acetyl-tryptophan has been covalently coupled to an amino-resin (aminoethylated Bio-Gel), and the resulting polymer reacted with a mixture of peptides in presence of 4,6-dinitrophenyl-1,3-disulfenyl chloride in glacial acetic acid. Selective covalent binding of tryptophan peptides has been checked.

Chromatography, Gel↗
PubMed1975

Labelling of the indole nucleus of tryptophan at the 2-position.

Tryptophan may be converted in high yields to the 2-hydroxy-derivative by reaction of sulfenyl halides and subsequent hydrolysis in 20% acetic acid at high temperature of the 2-thioaryl-compound. 2-Thiol-tryptophan and related compounds have been obtained by reduction of the tryptophan dimers obtained by reaction with sulfur dicholoride (S2Cl2). Novel methods of isotopic labelling the indole moiety at the 2-position have been also developed. 2-Thioaryl-indole derivatives with a propionic acid side chain at the 3-position are converted by one equivalent of N-bromosuccinimide in bicarbonate solution to lactones, which upon reduction with NaBH4 (or NaBD4 or NaBT4) give indole-propionic acid derivatives (or 2-labelled compounds thereof). The incorporation of deuterium or tritium was approximately 80%.

Bromosuccinimide↗
PubMed1975

Studies on the function of tryptophan-108 on lysozyme.

Chemical studies of selective modification of Trp-108 of lysozyme gave ambiguous results concerning its function on the catalytic activity, since the oxyndole derivative obtained with N-bromosuccinimide is inactive, whereas the kynurenine derivative obtained by oxidation with ozone is fully active. In order to explain this discrepancy, lysozyme has been modified with 2-nitro-4-carboxyphenylsulfenyl chloride (NCPS-Cl). This reagent reacts with the indole ring of tryptophan giving a 2-thioaryl-derivative. By chromatographic fractionation of the reaction mixture, a lysozyme derivative was isolated, that by sequence studies was proved to be modified only at Trp-108 retaining 10% of the lytic activity. Physico-chemical as well as kinetic studies indicate that the large decrease in activity following modification could be related to minor effects in the microenvironment of the active site, with a concomitant modification of the ionization constants of the groups involved in catalysis.

Acetylglucosamine↗