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L Politi

Publications and source records attributed to L Politi.

At least 37 records · Page 2Linked to original sources
PubMed1994

The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium.

The complete amino acid sequence of glutamate dehydrogenase from the archaebacterium Pyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium, Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992), Eur. J. Biochem. 203, 81-87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase from Pyrococcus furiosus with that of S. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Amino Acid Sequence↗
PubMed1993

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus: studies on thermal and guanidine-dependent inactivation.

The hexameric NAD(P)-dependent glutamate dehydrogenase isolated from the thermoacidophilic archaebacterium Sulfolobus solfataricus shows a remarkable thermal stability which is strictly dependent on protein concentration (half-life at 95 degrees C is 0.25 h and 0.5 h at 0.4 and 0.8 mg/ml, respectively). Temperature-dependent inactivation of the enzyme is apparently irreversible; this process is accompanied by a progressive increase in hydrophobic surface area which leads to protein precipitation. 3 M GdnHCl increases the half-life of the enzyme at 90 degrees C and 0.2 mg/ml 6-fold. The hexamer is the only soluble molecular species revealed by glutaraldehyde fixation after thermal inactivation. Lyotropic salts strongly affect the enzyme thermal stability: the half-life at 90 degrees C and 0.2 mg/ml protein concentration increases more than 6-fold in the presence of 0.4 M Na2SO4 and decreases 4-fold in the presence of 0.4 M NaSCN. The maximum protein thermal stability is observed around the isoelectric pH, between pH 5.2 and pH 6.8. Guanidine-dependent inactivation of the enzyme at 20 degrees C is irreversible above 1.5 M GdnHCl. The decline in percentage of reactivation closely parallels the structural changes detected by fluorescence and the loss of hexameric structure accompanied by the dissociation to monomers, as indicated by glutaraldehyde fixation.

Enzyme Activation↗
PubMed1992

The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium. Is the presence of N-epsilon-methyllysine related to thermostability?

The complete amino acid sequence of glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage by trypsin, cyanogen bromide, Staphylococcus aureus V8 protease and pepsin. The enzyme subunit is composed of 421 amino acid residues yielding a molecular mass of 46.078 kDa. The presence of N-epsilon-methyllysine in six positions of the sequence was observed. Comparison of the sequence of glutamate dehydrogenase from S. solfataricus with the other known primary structures of the corresponding enzyme from different sources, gives an overall identity of 9.2% and shows a symmetrical evolutionary distance of this archaebacterial protein from the two groups of vertebrate on one side and eubacterial and low eucaryote enzymes on the other side. The occurrence of specific substitutions and a possible role for N-epsilon-methylation of lysine residues are discussed in view of current hypotheses on the molecular basis of thermal adaptation of proteins.

Amino Acid Sequence↗
PubMed1991

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pI of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100 degrees C was 12 h), totally independent of enzyme concentration. P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P. furiosus.

Amino Acids↗
PubMed1991

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80 C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.

Archaea↗
PubMed1991

Proteoglycan synthesis in cultures of murine retinal neurons and photoreceptors.

1. In recent years, a number of histochemical and immunocytochemical studies have suggested that proteoglycans, particularly those in the interphotoreceptor matrix, exhibit altered distributions in several murine models for retinal degenerations. We are using a cell culture system to characterize the proteoglycans synthesized by neurons and photoreceptors derived from mouse retina, with the long-term goal of analyzing their role in retinal degenerations. 2. In this study we describe initial studies using cells derived from the retinas of normal mice. Cultures of retinal neurons and photoreceptors, which were free of glial, epithelia, or endothelial cells, were labeled with 3H-glucosamine and 35SO4. Proteoglycans isolated from the medium and cell layer were analyzed on the basis of charge, relative hydrodynamic size, and glycosaminoglycan content. 3. The studies indicate that the cultures actively synthesize proteoglycans. The medium contained predominantly chondroitin sulfate/dermatan sulfate, while the cell layer had a higher proportion of heparan sulfate, indicating a differential distribution between the two compartments.

Animals↗
PubMed1989

Oxalate, phosphate and sulphate determination in serum and urine by ion chromatography.

A rapid method for the determination of phosphate, sulphate and oxalate in serum by ion chromatography is described. Serum is deproteinized through a Centrifree filter by centrifugation and the ultrafiltrate directly injected into an ion chromatograph equipped with an anion exchange column and a conductivity detector. By this procedure the sample is not diluted and even small amounts of oxalate in biological fluids can be detected. Mean serum concentrations found in healthy individuals are: phosphate 1.07 mmol/l; sulphate 0.35 mmol/l; oxalate 21.02 mumols/l. Phosphate, sulphate and oxalate contents were also determined in urine from healthy individuals. Values found in serum and urine are in good agreement with those previously reported.

Chromatography, Ion Exchange↗
PubMed1989

Perfusion of uremic blood ultrafiltrate on uncoated charcoal.

A granular uncoated charcoal removes from uremic blood ultrafiltrates many chemical species that are not removed by dialysis. Charcoal treatment dramatically improves the general condition of the patients and normalizes their blood pressure. To obtain a rapid depuration, the initial treatment should be intensive (at least 16 daily treatments) and the effects prolonged over time by once-a-week charcoal treatment between two standard hemodialyses. Biogel P2 chromatography documents well all the events of a depurative treatment that cannot be monitored by hematochemical analyses.

Charcoal↗
PubMed1988

Functional residues at the active site of horse liver phosphopantothenoylcysteine decarboxylase.

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) is rapidly inactivated by N-acetoacetylation with diketene following a pseudo-first-order kinetics: the presence of substrate quantitatively protects against this inactivation. Histidine photo-oxidation with methylene blue or rose bengal brings about the total loss of activity. These results indicate the presence of functional lysyl and histidyl groups at the active site of the enzyme. The substrate sulphydryl group is essential for enzyme activity. Enzymatic decarboxylation is proposed to result from a combined action of the keto group of the enzyme-bound pyruvate protonated by an essential histidine and a protonated amino group of a lysine.

Animals↗
PubMed1988

Selective failure of long-term survival of isolated photoreceptors from both homozygous and heterozygous rd (retinal degeneration) mice.

Retinas from homozygous rdle/rdle and heterozygous rdle/++ C57BL/6J mice were dissected and dissociated on postnatal day 2, when they are still essentially indistinguishable. The resulting cell suspensions were seeded on highly adhesive substrata, to which the cells attach as individual units, and grown in vitro for 2 weeks in serum-free, chemically defined media. The behavior of neurons and photoreceptors in vitro was investigated with several techniques; essentially no differences were found between rdle/rdle and rdle/++ cells. Three distinctive cell types could be recognized in cultures of both genotypes towards the end of the first week in vitro: process-free cells, multipolar neurons and rod photoreceptors. There were similarities between rdle/rdle and rdle/++ cultures in the number and morphology of photoreceptor cells, to include the presence of a cilium and a short neurite terminating in a spherule-like body. Moreover, in cultures of both genotypes, only photoreceptors showed opsin immunoreactivity and the antigen recognized by the rod-specific monoclonal antibody RET-P1. Biochemical and autoradiographic studies demonstrated that rdle/rdle and rdle/++ cells also showed similar uptakes of the putative amino acid neurotransmitters glutamate and aspartate (associated with most of the photoreceptors and only some neurons), and gamma-aminobutyric acid (associated with neurons but absent in photoreceptors). Thus, according to several parameters, the properties shown by photoreceptor cells were similar in rdle/rdle and rdle/++ cultures during the first week in vitro. Massive photoreceptor cell death was observed in both genotypes during the second week in vitro, coinciding with the time when photoreceptor degeneration occurs in vivo in rd/rd, but not in rd/+ retinas. Photoreceptor death in culture appeared to be specific, since approx. 80% of the non-photoreceptor neurons survived normally during the period when photoreceptor degeneration took place. Several reports from the literature suggest that the period around postnatal days 8-10 represents a critical stage for rd/rd photoreceptors, since they survive until this time but degenerate thereafter. Genetically normal photoreceptors apparently undergo a comparable crisis during maintenance in primary culture, suggesting the involvement of cell-cell contacts and/or retina-derived environmental signals in the survival or rod visual cells.

Amino Acids↗
PubMed1987

Covalently bound pyruvate in phosphopantothenoylcysteine decarboxylase from horse liver.

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) incorporates nonexchangeable tritium from borotritide with a decrease of the activity. Substrate prevents both tritium incorporation and the decrease in activity. Acid and base hydrolysis of the tritiated protein releases labeled lactate identified by high-voltage paper electrophoresis, paper chromatography and silicic acid chromatography. These results indicate the presence of pyruvate covalently bound through an ester linkage to phosphopantothenoylcysteine decarboxylase which is then another example of a mammalian enzyme in which pyruvate is involved in a catalytic activity.

Amino Acid Transport Systems, Neutral↗
PubMed1987

Selective destruction of photoreceptor cells by anti-opsin antibodies.

This article reports that cultured photoreceptor cells are selectively vulnerable to complement-mediated lysis by antiopsin antisera. The study has been carried out using a culture system that permits the growth of embryonic retinal neurons and photoreceptor cells in the absence of glial, pigment epithelial, connective, or endothelial cell contamination. Exposure of these cultures to an antiopsin antiserum in the presence of complement results in the lysis of 85% of the photoreceptor cells, without extensive loss of neurons. Photoreceptor lysis by the antibody is dose-dependent and specific, as demonstrated by the ability to block photoreceptor lysis by preincubation of the antiopsin antiserum with purified rhodopsin. Photoreceptor sensitivity to lysis by these antibodies develops in vitro in parallel with the appearance of immunocytochemically detectable opsin. Multipolar neurons (which do not contain opsin) are not affected by the treatment, as shown by microscopic analysis and determination of neuronal "markers" such as choline acetyltransferase and GABA high affinity uptake. These results show that antiopsin antibodies can cause selective photoreceptor degeneration. Moreover, this in vitro system appears useful as a bioassay to test a variety of possible mechanisms of retinal cell destruction which may be important in vivo. The results suggest also a mechanism for the generation of enriched retinal neuronal cultures by selective lysis of photoreceptor cells.

Animals↗
PubMed1986

[Role and limits of diagnostic imaging in the staging of lung cancer].

A total of 474 histologically proved Lung Cancers (LC) were evaluated by Conventional Radiology (CR) and Computed Tomography (CT) in order to assess the role of these two diagnostic modalities in the staging of LC. In 196/474 LC it was possible also to refer, for the evaluation of the diagnostic reliability, to the surgical control. The CR features of LC both at stage III (13% of the global series) and at stage I-peripheral T1 (16% of the global series) presented very high positive and negative predictive values (90% and 95%, retrospectively); in these cases it was considered useless to perform CT. The CT grading showed a high negative predictive value in excluding stage III caused respectively by grade T3 (91.5%) and grade N2 (93.5%). As to the staging, the CT assessment of the stage I and II showed a high predictive value (91.5%). On the other hand, the CT assessment of stage III presented an unsatisfactory predictive value (71%), due to the low predictive value in grade T3 (71.5%), poor in grade N2 (57.5%). The increase of the value threshold mediastinal adenopathies from 1 to 2 cm, leads to a great improvement of this predictive value (92%). The CT reliability was matched with the two different surgical "philosophies" (non aggressive or aggressive); surgery was excluded or performed according to the presence of omo-lateral mediastinal adenopathy. A correct advice to perform surgery was achieved in 33.5% (non aggressive "philosophy") and 43.8% (aggressive "philosophy") of cases; surgery was correctly excluded respectively in 49.6% and 45.9%. The resort to mediastinoscopy was advised in 7.8% and 1.3% of the cases respectively. In both "philosophies" the error of under-staging was lower (2.5%), than that of over-staging (6.6%).

Diagnostic Errors↗
PubMed1985

Chromatographic evaluation of perfusion on charcoal in uraemia.

Chromatography on Bio-Gel P-2 and high-performance liquid chromatography on RP-18 columns monitored with UV, fluorescence and electrochemical detectors have been used to evaluate the efficiency of granular, uncoated, active charcoal to remove from the ultrafiltrates of uraemic patients those organic substances accumulated in the blood that are not easily removed by dialysis. Chromatography on Bio-Gel P-2 and high-performance liquid chromatography on RP-18 columns carried out isocratically and monitored with an electrochemical detector seem very useful for clinical investigation as they increase the information obtained from routine haematochemical analyses such as blood urea nitrogen, creatinine, uric acid and electrolytes (calcium, phosphorus, sodium and potassium).

Charcoal↗
PubMed1981

Evaluation of blood toxicity in chronic uremia by an improved chromatographic method.

Biogel P2 gel chromatography of ultrafiltrate from chronic uremic patients leads to better resolution of the compounds present than so far reported in the literature. Reproducibility is good. Toxicity, evaluated by cell cultures, was detected in only one chromatographic fraction. Further purification of this fraction with HPLC on a RP18 column revealed two toxic fractions, to the better defined of which a polyol structure was ascribed.

Animals↗