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V Bocchini

Publications and source records attributed to V Bocchini.

At least 73 records · Page 4Linked to original sources
PubMed1989

Effect of propan-2-ol on enzymic and structural properties of elongation factor G.

Elongation factor G (EF-G) can support a GTPase activity in vitro even in the absence of ribosomes when propan-2-ol is present [GTPasep; De Vendittis, Masullo & Bocchini (1986) J. Biol. Chem. 261, 4445-4450]. In the present work the GTPasep activity of EF-G was further studied by investigating (i) the effect of ionic environment on GTPasep and (ii) the influence of propan-2-ol on the molecular structure of EF-G as determined by fluorescence and c.d. measurements. In the presence of 1-300 mM univalent cations (M+) alone, no detectable GTPasep activity was measured; however, in the presence of 1 mM-Mg2+ a considerable stimulation was observed at 40 mM-Li+ or 75 mM-NH4+. Among bivalent cations (M2+), 1 mM-Sr2+, 2-5 mM-Ca2+ and 1 mM-Ba2+ were the most effective, but, in the presence of 75 mM-NH4+, Mg2+ and Mn2+ became the most efficient, whereas the stimulation by other M2+ species was considerably decreased. C.d. measurements showed that the alcohol increased the mean molar residue ellipticity of EF-G at 285 nm, but not at 220 nm. As estimated from fluorescence measurements, in the presence of 20% (v/v) propan-2-ol the value of the dissociation constant of the complex formed between EF-G and 8-anilino-1-naphthalene-sulphonate decreased from 8 to 5 microM; similarly, the number of binding sites on EF-G for the fluorescent probe decreased from 13 to 6. Finally, the alcohol enhanced the quenching of the intrinsic fluorescence of EF-G caused by either acrylamide or KI. The data support the hypothesis that propan-2-ol induces moderate conformational changes of EF-G that make the catalytic centre accessible to the substrate even in the absence of ribosomes. Kinetics of GTPasep studied at different temperatures did not reveal additional structural changes of EF-G occurring with time or temperature.

1-Propanol↗
PubMed1988

Latex beads phagocytosis capacity and ecto-NAD+ glycohydrolase activity of rat brain microglia cells in vitro.

Conditions are described which allow the preparation in vitro of pure (greater than 95%) microglial cell cultures isolated from newborn rat brain. Such ameboid cells cultivated in vitro can efficiently phagocytize opsonized latex beads and are capable of ingesting more (100-200 beads of 1.1 micron diameter per cell) and larger (6.4 microns) particles than other nerve cells, such as oligodendrocytes and astroglia. The microglial cells also show an important ecto-NAD+ glycohydrolase activity which is characteristic of phagocytic cells. We noted that the phagocytic capacity and ecto-NAD+ glycohydrolase of these cells were correlated and increased notably during the in vitro culture. Microglia cultivated in vitro appear to be a good model to study the activation of phagocytic properties in the central nervous system and corresponding modulation by natural or pharmacological immunomodulators.

Animals↗
PubMed1988

Phagocytosis of polystyrene latex beads by rat brain microglia cell cultures is increased by treatment with gangliosides.

Cultures of microglial cells were prepared from primary rat brain cultures of astrocytes. Microglia appeared under two main forms: ameboid and ramified. Only the former were capable of ingesting opsonized latex particles of 1 and 6 micron; while the latter, which appeared later in culture, were not capable of phagocytosis. When gangliosides (10(-6) M), which are known for their neuronotrophic capacity in vitro, were added to the growth medium (for 20 min) they stimulated the phagocytosis of ameboid microglia 2-fold.

Animals↗
PubMed1986

Characterization of the elongation factors from calf brain. 1. Purification, molecular and immunological properties.

This work describes a method for the purification of the elongation factors (EF) from calf brain. The elongation factor responsible for the binding of aminoacyl-tRNA to the ribosome is found in this organ as a light form (EF-1 alpha) and as a component of heavy, polydispersed aggregates (EF-1H). EF-1 beta, the factor enhancing the EF-1 alpha GDP/GTP exchange, is part of EF-1H and of smaller aggregates. The fraction of EF-1 alpha and EF-1 beta not associated with EF-1H, and EF-2 have been purified to homogeneity after several chromatographic steps. EF-1H consists of many proteins; among them, EF-1 alpha, EF-1 beta and an EF-1 gamma-like protein represent three of the major components. This conclusively shows that EF-1H from calf brain is not a polydispersed aggregate of only EF-1 alpha. EF-1 beta has also been purified to homogeneity from EF-1H. The property of EF-1 beta to aggregate with other proteins suggests that this factor plays an important role in the organization of EF-1H. The relative molecular mass of the purified factors have been determined as: EF-1 alpha, 50,000; EF-1 beta, 30,000; the EF-1 gamma-like component, 49,000; EF-2, 85,000. Some cross-reactivity with the antibodies against the prokaryotic counterparts has been shown for EF-1 alpha, EF-1 beta and EF-2 by functional and immuno-precipitation methods, suggesting the existence of structural homologies.

Animals↗
PubMed1986

The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes.

In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium.

1-Propanol↗
PubMed1984

Purification and characterization of four different alkaline phosphatases from Spirographis spallanzanii.

Alkaline phosphatases (APs) present in Spirographis spallanzanii were extracted by water homogenization of butanol treatment. The enzymes solubilized as above were purified by acetone fractionation and then by DEAE-cellulose and Sephadex G-200 chromatography; separation of four AP forms was achieved, which were characterized by studying their molecular and catalytic properties. The various APs differ in molecular weight, electrophoretic mobility, optimum pH, but show similar Km values and substrate inhibition pattern. Kinetic studies carried out with several inhibitors show, in particular, the existence in these APs of a second binding site and suggest a possible role for them in the metabolism of phosphoric esters of the sugars.

Alkaline Phosphatase↗
PubMed1984

[Radioimmunoassay of high specificity for vincristine].

The authors set up a simple and fairly rapid radioimmunoassay for vincristine, that shows a good sensitivity, precision and specificity. In particular, the higher specificity in comparison with other similar dosage techniques is likely due to the marked specificity of the used anti-vincristine serum, which displays a low interference even by molecules with a vincristine-like structure. Therefore, the suggested radioimmunoassay technique seems quite suitable for studying vincristine pharmacokinetics and for monitoring blood levels of this alkaloid in treated patients.

Evaluation Studies as Topic↗
PubMed1984

Purification of S-2-hydroxyacylglutathione hydrolase (Glyoxalase II) from calf brain.

S-2-Hydroxyacylglutathione hydrolase (Glyoxalase II) from calf brain has been purified 8333-times compared to 65,000 g supernatant of brain homogenate. The purification procedure employs Affi-Gel blue and preparative isoelectric focussing and offers a suitable method for the preparation of highly purified enzyme. Calf brain Glyoxalase II is a basic protein with a pl of 7.63 determined by isoelectric focusing. An evaluation of the relative molecular mass by gel filtration gave a value of about 23,000. During the purification procedure a constant Km value of about 0.325 mM was observed. A turnover number of 16,100 min-1 was calculated for the purified enzyme.

Animals↗
PubMed1983

Superoxide dismutase activity in the skin of rats irradiated by He-Ne laser.

The activity of the enzyme superoxide dismutase has been measured in the skin of rats irradiated by a low-power He-Ne laser. The irradiation was performed at the doses of 4, 8 and 20 J/cm2 in a single or repeated treatment. The increase in activity of superoxide dismutase in the skin of irradiated animals was not statistically significant.

Animals↗
PubMed1983

Liver regeneration after atypical hepatectomy in the rat. A comparison of CO2 laser with scalpel and electrical diathermy.

Rats were subjected to partial hepatic lobectomy with removal of about two thirds of the liver either by scalpel, by CO2 laser, or by electrical diathermy. Macroscopic examinations of livers at various days after surgery indicated that CO2 laser surgery results in minor tissue damage, reduced complications from inflammation and infections, and faster healing compared to electrical diathermy, but it is less satisfactory than the scalpel with respect to all these parameters. Rat liver regeneration was followed at the molecular level by measuring the changes of protein biosynthesis activity in the liver, as monitored by modifications of ribosome organization. Activation of the protein biosynthesis process started 1 day after surgery when hepatectomy was performed by scalpel, while 2 days were required when CO2 laser or electrical diathermy were used instead.

Animals↗
PubMed1983

Behavior of some enzymatic activities in the serum of rabbits after tissue damage by CO2 laser radiation.

A comparative evaluation of the biochemical damage observed after tissue injury by high-power CO2 laser, scalpel, and electrical diathermy has been made in rabbits. The tissue injury consisted of surgery performed by making two perpendicular linear cuts on the muscle of the posterior thigh. At different times after surgery, enzymatic levels of creatine kinase, aspartate amino transferase, and lactate dehydrogenase were determined in the serum. Only creatine kinase showed an increase, and it was lower in both extent and dispersion in the animals treated with CO2 laser. The results suggest that CO2 laser radiation caused damage to muscular tissue which was moderately lower compared to that from scalpel or electrical diathermy.

Animals↗
PubMed1982

DHT-receptor in cultured human fibroblasts: binding study in a family with androgen insensitivity (complete testicular feminisation).

3H-DHT binding was examined in cultured skin fibroblasts from a patient with complete testicular feminisation (CTF), from his heterozygote mother, and his clinically normal sister, who menstruated normally. Binding parameters were: Bmax less than 1 fmol/mg protein and KD unmeasurable in CTF; Bmax = 24 fmol/mg protein and KD = 3.63 X 10(-9) mol/l in the mother; and Bmax = 46 fmol/mg protein and KD = 3.7 X 10(-9) mol/l in the sister. Five cultures obtained from genital and non-genital skin of normal male and female subjects were used as controls, in which Bmax ranged from 37 to 62 fmol/mg and KD from 2.0 to 3.6 X 10(-9) mol/l. The considerable reduction of Bmax in the obligate heterozygote and the normal binding capacity in the sister, a probable heterozygote, suggests that it may be possible to use the DHT-receptor assay to identify carriers in families with androgen resistance.

Adolescent↗