Biomedical subjects

F Dallocchio

Publications and source records attributed to F Dallocchio.

At least 37 records · Page 2Linked to original sources

PubMed1991

Allosteric inhibition of the water-soluble C-terminal fragment of Sendai virus neuraminidase.

Trypsin solubilized hemagglutinin-neuraminidase of Sendai virus (cHN) displays michaelian kinetics, with native fetuin as substrate, at 37 degrees C. Vmax and Km values are only marginally altered, as compared to intact viral neuraminidase. At lower temperatures, cHN follows non-michaelian kinetics, with marked substrate inhibition at 4 degrees C. With denaturated fetuin, michaelian kinetics are observed in all conditions, while asialo fetuin was an uncompetitive inhibitor of cHN, with native fetuin or sialyl lactose as substrates. These results can be explained assuming that the protein moiety of fetuin acts as an allosteric inhibitor of cHN.

Allosteric Site↗

PubMed1991

Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.

Animals↗

PubMed1990

Myelin basic protein inhibits the calcium response to phytohaemagglutinin in human lymphocytes.

Myelin Basic Protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation by phytohaemagglutinin. Pre-treatment of human lymphocytes with myelin basic protein results in a lower rising of cytosolic concentration of free calcium after stimulation with phytohaemagglutinin. This effect is dependent on myelin basic protein concentration and on the preincubation time of the protein with the cells. It is not due to a interaction between myelin basic protein and phytohaemagglutinin, but appears to be a consequence of the binding of the protein to the cell surface. The reduction of the rise of cytosolic calcium induced by phytohaemagglutinin is specific for the myelin basic protein because other proteins like albumin and protamine have no effect.

Calcium↗

PubMed1990

Effect of 17 beta-estradiol on calcium response to phytohaemagglutinin in human lymphocytes.

Pre-treatment of human lymphocytes with 17 beta-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17 beta-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17 beta-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17 beta-estradiol, since the alpha isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5 alpha-androstan) have no effect. Since the effect of the 17 beta-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.

Calcium↗

PubMed1989

Inactivation of skeletal-muscle UDP-glucose pyrophosphorylase by reaction with carboxylate-directed reagents.

Skeletal-muscle UDP-glucose pyrophosphorylase is inactivated by reaction with 2-ethoxy-N-(ethoxy-carbonyl)-1,2-dihydroquinoline (EEDQ) and 1-(3-dimethylaminopropyl-3-ethylcarbodi-imide (EDAC), two reagents specific for carboxylate groups. The former reagent is a more effective inactivator than EDAC. Although no evidence of reversible enzyme-reagent complexes of the affinity-labelling type was obtained by kinetic analysis of the inactivation, the selective protection of UDP-glucose pyrophosphorylase activity against inactivation by EEDQ in the presence of uridine substrates is indicative of an active-site-directed effect. The results are consistent with the hypothesis that EEDQ modifies a single carboxylate group located in a hydrophobic domain close to the substrate-binding site, leading to enzyme inactivation. In contrast, the reaction between UDP-glucose pyrophosphorylase and EDAC appears to involve a different region of the enzyme.

Animals↗

PubMed1989

Effect of 17 beta-estradiol on partition behavior of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases.

Preincubation with 1 nM 17 beta-estradiol changes the partition behaviour of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases. Erythrocyte partition decreases reaching a minimum after 5 min of incubation, then slowly increases returning to starting values after 30 min. The effect is specifically induced by the isomer of estradiol, whereas the alpha isomer and other steroid hormones (progesterone, testosterone and 5 alpha-dihydrotestosterone) are uneffective. The effect of the hormone can be suppressed by treatment of erythrocytes with either N-ethyl maleimide, sodium vanadate, or ouabain, or carrying out incubations in potassium-free buffer. These data suggest that the effect of estradiol on phase partition of erythrocytes is mediated by the (Na,K)-ATPase.

Dextrans↗

PubMed1988

Inhibition of activity and quenching of intrinsic fluorescence of transglutaminase by acrylamide are independent events.

Addition of low concentrations of acrylamide to the assay mixture of erythrocyte transglutaminase leads to a strong inhibition of the enzyme with a mechanism consistent with a non-competitive inhibition against both substrates. For the quenching of the intrinsic fluorescence of the protein, much higher concentrations of acrylamide are required so that both phenomena appear independent of each other. In this particular case, therefore, acrylamide can be employed to obtain information on ligand-triggered conformational changes.

Acrylamide↗

PubMed1986

Myelin basic protein inhibits formyl-peptide induced chemotaxis in human neutrophils.

Myelin basic protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation. Pre-treatment of human neutrophils with myelin basic protein selectively inhibits the formyl-peptide-induced chemotaxis, without affecting chemotaxis evoked by casein and activated serum. Furthermore, both the degranulation and superoxide anion production stimulated by the chemotactic peptide are not modified by the prior treatment of the neutrophils with myelin basic protein.

Calcium↗

PubMed1986

A rapid method for purification of myelin basic protein.

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.

Acetates↗

PubMed1986

Inactivation of skeletal glycogen synthetase by diethylpyrocarbonate.

Glycogen synthetase from skeletal muscle is rapidly inactivated by DEPC. In the presence of the substrate UDPG only 50% of the enzyme activity is lost. The concomitant addition of both UDPG and the allosteric activator glucose-6-phosphate almost completely prevents the inactivation by DEPC. Since glucose-6-phosphate alone does not prevent the inactivation by DEPC, it is concluded that it is effective through a potentiation of the effects of UDPG, possibly through a conformational change of the enzyme.

Animals↗

PubMed1985

Half-site reactivity in 6-phosphogluconate dehydrogenase from human erythrocytes.

6-Phosphogluconate dehydrogenase from human erythrocytes was purified by an improved procedure. Binding studies showed that the dimeric enzyme binds 2 mol of NADP+/mol but only 1 mol of NADPH/mol, and that the bindings of oxidized and reduced coenzyme are mutually exclusive. From initial-rate kinetics and inhibition studies, a sequential random-order mechanism is proposed. Double-reciprocal plots with NADP+ as varied substrate show a downward curvature, indicating a negative co-operativity. We suggest that the negative co-operativity observed kinetically is a result of the half-site reactivity for the NADPH. The different binding stoichiometries for NADP+ and NADPH generate a non-linear relationship between the apparent dissociation constant for the NADPH and the concentrations of the NADP+, resulting in a regulatory mechanism highly sensitive to the changes in the NADP+/NADPH ratio.

Binding Sites↗

PubMed1985

Hormonal sensitivity of adenylate cyclase from human endometrium: modulation by estradiol.

In human endometria, a membrane-bound adenylate cyclase is present, which is recovered in high yield in a low-speed particulate fraction. Neither the specific activity of the enzyme nor the response to modifiers that act through the regulatory subunit of the complex, are modified during the proliferative or secretory phase of the cycle. Surprisingly, we found that in vitro treatment of secretory endometrial membranes with 17 beta-estradiol stimulates 3- to 4-fold the activity of adenylate cyclase. However this response does not occur on proliferative membranes. The activation by estradiol is independent of the presence of guanylylimidodiphosphate and is additive to that of the nucleotide. Possibly, as the consequence of the phenomenon, the concentration of cyclic AMP is significantly higher in curretage samples obtained from patients during the secretory rather than in the proliferative phase of the cycle. To our knowledge this is the first evidence of a target-cell membrane-directed effect of sex steroids in humans.

Adenylyl Cyclases↗

PubMed1985

The NADP binding site on rabbit muscle aldolase.

Rabbit muscle aldolase binds NADPH with a 1:1 stoichiometry and with a dissociation constant 18 microM. Three sites of the dinucleotide are involved in the binding: the adenosyl diphosphate moiety, the nicotinamide-ribose, and the nicotinamide ring. These data show the existence of a specific dinucleotide binding site in the aldolase molecule.

Animals↗

PubMed1984

Purification of muscle uridine diphosphoglucose pyrophosphorylase by hydrophobic chromatography.

Uridine diphosphoglucose pyrophosphorylase was purified 2500-fold from rabbit skeletal muscle with a total recovery of 35% of the initial activity. The present procedure was made possible by an extensive use of hydrophobic chromatography. Purified pyrophosphorylase had a specific activity of 500 mumol/min/mg of protein and was homogeneous by chromatographic and electrophoretic criteria. The enzyme appears to be composed of eight subunits of 53,000 molecular weight each.

Amino Acids↗

PubMed1983

Non-Michaelian kinetics of rabbit muscle uridine diphosphoglucose pyrophosphorylase.

The kinetic properties of rabbit muscle uridine diphosphoglucose (UDP-Glc) pyrophosphorylase have been studied, in both directions, with respect to substrate saturation, product inhibition, and cation requirement for activity. The results demonstrate that UDP-Glc pyrophosphorylase is a non-Michaelian enzyme: the synthetic reaction is characterized by a marked inhibition by glucose-1-phosphate (at concentrations higher than 0.3 mM) and by an hyperbolic saturation for UTP. In the reverse reaction, instead, the saturation function for UDP-Glc is hyperbolic and that for inorganic pyrophosphate is sigmoid, with a high Hill coefficient of (nH) 2.5. The study of the metal requirement indicates a distinctive ability of cations to stimulate the reactions of synthesis and degradation of the sugar nucleotide and a different stoichiometry of the metal chelates involved. The reaction mechanism is of the ordered-sequential type and the data of product inhibition allowed the identification of glucose-1-phosphate as the first substrate bound and UDP-Glc as the last product released. The inhibition pattern by UDP-Glc gives evidence for cooperativity also in the binding of this molecule.

Animals↗

PubMed1982

Non-enzymic protein phosphorylation. Phosphorylation of 6-phosphogluconate dehydrogenase by acyl phosphates.

Incubation of 6-phosphogluconate dehydrogenase from Candida utilis with either acetyl phosphate, 1,3-diphosphoglycerate or carbamoyl phosphate results in the phosphorylation of the protein. The binding of one phosphate residue per enzyme subunit does not affect significantly the kinetic properties, but makes the enzyme less reactive toward thiol reagents, trypsin and pyridoxal 5'-phosphate. We suggest indicate that: (1) 6-phosphogluconate dehydrogenase from C. utilis is phosphorylated non-enzymically by physiological acyl phosphates and (2) the phosphorylation of the enzyme modifies the rate of protein inactivation.

Amino Acids↗

PubMed1981

Substrate-induced intramolecular proton transfer in 6-phosphogluconate dehydrogenase from Candida utilis.

Formation of binary complex between 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) from Candida utilis and 6-phosphogluconate was investigated by means of ultraviolet difference spectroscopy. The formation of the enzyme-substrate complex induces in the difference spectrum a positive peak the wavelength and extinction coefficient of which agree well with a tyrosine ionization. Titrimetric studies indicate that the formation of the binary complex is not coupled to a proton release from the protein. These data support an intramolecular proton transfer from a tyrosine to other functional group. This proton transfer could be correlated to the conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

Candida↗

PubMed1981

Effect of the substrate on the binding of coenzyme and coenzyme analogues to 6-phosphogluconate dehydrogenase from Candida utilis.

6-Phosphogluconate dehydrogenase from Candida utilis is a dimeric enzyme with apparently identical subunits. Two coenzyme analogues, periodate-oxidized NADP+ and 3-amino pyridine adenine dinucleotide phosphate, bind to the protein with Kdiss of 42 microM and 6.2 microM, respectively. NADPH binds to the enzyme with a Kdiss of 0.42 microM. Both coenzyme and coenzyme analogues show "all-of-the sites reactivity." In the presence of the substrate 6-phosphogluconate, the Kdiss of the coenzyme analogues is lowered to 3.5 microM and 2.4 microM, respectively, whereas the Kdiss of NADPH is increased to 2.7 microM. Coenzyme analogues, but not NADPH, show an half of-the sites reactivity in the presence of 6-phosphogluconate. The enzymatic activity is inhibited by high NADP+ concentrations in presence of low concentrations of substrate, whereas at high substrate concentrations no inhibition by NADP+ is showed. These data suggest that: 1) the enzyme exhibits an half-of-the-sites reactivity during the catalytic cycle; 2) the recycle of the enzyme occurs mainly through an abortive ternary complex enzyme-substrate-NADPH.

Candida↗