Biomedical subjects

G Lupidi

Publications and source records attributed to G Lupidi.

At least 19 recordsLinked to original sources

PubMed2002

Interaction of Hsp90 with 20S proteasome: thermodynamic and kinetic characterization.

The proteasome and heat shock proteins have been found in the centrosome. The evidence of their copurification reported by several studies suggests that they form stable complex. In addition, Hsp90 is involved in the loading of proteasome-generated antigenic peptides to the class I major histocompatibility complex. In this article, we report a detailed thermodynamic and kinetic characterization of the Hsp90-20S proteasome interaction, using a surface plasmon resonance technique. The modulation exerted by protons in solution has been investigated, and the results have been discussed, taking into account structural motifs characterizing the binding interface between the two macromolecules.

Animals↗

PubMed2002

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain.

The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

Alzheimer Disease↗

PubMed2002

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate.

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors--erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine--was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme-inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors--derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.

Adenosine Deaminase↗

PubMed2001

Structure--function relationships in bovine thymus 20S proteasome: a fluorimetric study.

The structure--function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle.

Animals↗

PubMed2001

Tryptophan environment in adenosine deaminase. I. Enzyme modification with N-bromosuccinimide in the presence of adenosine and EHNA analogues.

Adenosine deaminase from bovine cerebral hemisphere (white and gray matter) and spleen was treated with N-bromosuccinimide, a reagent known to oxidize selectively tryptophan residues in proteins. Spectrally observable tryptophan modification was accompanied by enzyme inactivation. Tsow graphics revealed that two Trps are essential for the activity of enzyme from both tissues. Enzyme inhibitors and substrate analogues, derivatives of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine, were able to protect Trp against modification, and this effect correlated in general with the enzyme activity protection. In the presence of adenosine deaza analogues (the noninhibitor tubercidin among them) only two Trps were modified in the fully inactivated enzyme. In the presence of EHNA and its deaza analogues, full inactivation of the enzyme was accompanied by the modification of four Trps. The obtained data confirm the previous hypothesis about the presence on the enzyme of different binding sites for adenosine and EHNA derivatives that are responsible for the different effects on the enzyme conformation elicited by the corresponding derivatives. Moreover, these data allow us to suggest that Trp residues, still unidentified by X-ray analysis, are essential for the functioning of the enzyme.

Adenine↗

PubMed2001

Adenosine deaminase: functional implications and different classes of inhibitors.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency disease (ADA-SCID), in which both B-cell and T-cell development is impaired. ADA-SCID has been the first disorder to be treated by gene therapy, using polyethylene glycol-modified bovine ADA (PEG-ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inhibitors have been designed and synthesized, classified as ground-state and transition-state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

Adenosine Deaminase↗

PubMed2001

Purification and partial characterization of glyoxalase I from bovine brain.

Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.

Animals↗

PubMed2001

Functional residues on the enzyme active site of glyoxalase I from bovine brain.

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.

Animals↗

PubMed2000

Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study.

The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.

Bacterial Proteins↗

PubMed2000

Isolation and characterization of bovine thymus multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC or proteasome) from bovine thymus was isolated and purified to homogeneity applying a protocol utilizing ion exchange and gel permeation chromatography as major purification tools. The purified complex shows molecular properties that are common for proteasomal molecules (high molecular mass, multisubunit organization, and multiple proteolytic activities) even though a peculiar subunit composition and the presence of specific regulatory mechanisms affecting the assembled proteolytic activities suggest a specialized function for this complex. Thymus proteasome is characterized by the presence of LMP2, LMP7, and LMP10 (MECL1) subunits, which replace the X, Y, and Z subunits. Since a similar complex was previously isolated in bovine spleen, it appears that the proteasomal population containing the LMP subunits is characteristic for organs involved in immune response. Both the thymus and spleen proteasomes are characterized by a marked efficiency in cleaving peptide bonds after branched-chain and aromatic amino acids, indicating that this proteasomal population is most likely involved in intracellular processing of class I antigenic peptides and is an example of an "in vivo" functioning immunoproteasome. However, in spite of several similarities, the complexes isolated from the two lymphoid organs do not show superimposable functional properties, which suggests the presence of organ-specific regulatory mechanisms affecting each of the proteolytic components assembled in the complex.

Animals↗

PubMed1999

Peroxynitrite-mediated oxidation of fibrinogen inhibits clot formation.

The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to inhibit 50% of fibrinogen clotting was 22 microM at 25 degrees C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO-, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite-modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite-mediated modification of fibrinogen.

Bilirubin↗

PubMed1998

Inhibition studies on membrane adenosine deaminase from human placenta.

The ecto form of adenosine deaminase isolated from human placental membrane was tested towards its sensitivity against adenosine deaminase inhibitors, such as aza and deaza analogues of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Ki values of the inhibitors observed were similar to these obtained for the small form of adenosine deaminase purified from human erythrocytes, indicating that the presence of the binding protein on placental adenosine deaminase does not produce alteration in the binding of these inhibitors on the enzyme active site. The inhibition rate of 2'-deoxycoformycin, one of the most potent ADA inhibitors is affected by the presence of the binding protein on human placental adenosine deaminase, that probably modulates the rearrangement of the active site produced by the binding with this tight-binding inhibitor.

Adenine↗

PubMed1997

Effect of mercuric ions on human erythrocytes. Relationships between hypotonic swelling and cell aggregation.

This study was undertaken to verify the hypothesis that the haemolytic effect of mercuric ions on human erythrocytes is strongly decreased under swelling conditions (relative to isotonic suspensions). In fact, interaction of Hg2+ with swollen erythrocytes yields a rapid and cooperative cell aggregation, a phenomenon that appears to prevent penetration of mercuric ions into the cells and, accordingly, to avoid any haemolytic effect induced by the Hg2+ entrance. Since in vivo erythrocytes undergo big shape changes (swelling being a kind of shape modification) related to mechanical or (in some animals) osmotic stresses, the reported observations turn out to be also of some relevance for the understanding of certain toxicological effects of mercuric ions.

4-Chloromercuribenzenesulfonate↗

PubMed1997

Human placenta cytidine deaminase: proton-linked enzyme activity and substrate binding.

In this work we focused our attention on some catalytic site requirements for human placenta cytidine deaminase (CDA). The effect of pH on substrate binding and catalysis was studied between pH 3.0 and pH 11.0. The results could be discussed postulating the presence of two classes of ionizable groups in the active site of CDA. The kinetic parameters pH-dependence has been discussed considering the presence of four zinc atoms per each enzyme tetramer. Furthermore fluorescence studies on the enzyme and on enzyme-inhibitor complexes, examined by using polar and non polar quenchers, allowed to define the substrate(inhibitor)-dependent accessibility of the tryptophan-containing pocket on each CDA monomer.

Binding Sites↗

PubMed1997

Functional residues at the active site of bovine brain adenosine deaminase.

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first-order kinetics with a second-order rate constant of 8.9 10(-3) (+/- 1.8 10(-3)) M-1 min-1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground- and transition-state analogs to the enzyme.

Adenosine Deaminase↗

PubMed1996

Acid-stable serine proteinase inhibitors in the urine of Alzheimer disease subjects.

A comparative study of the levels of acid-stable proteinase inhibitors (kallikrein and trypsin inhibitors) in the urine of healthy and Alzheimer subjects, of both sexes, has been performed. A preliminary characterization of the purified inhibitors indicates that the urinary antitryptic activity is accounted for by the presence of the well known Urinary Trypsin Inhibitor (UTI) while an apparently new molecule appears to be responsible for the antikallikrein activity. The urinary levels of kallikrein inhibitors are very similar in healthy and sick subjects while the levels of trypsin inhibitors appear significatively increased in Alzheimer subjects of both sexes. The data presented here support the hypothesis that unpaired proteolytic processes could be involved in the pathogenesis of Alzheimer's disease and suggest that the levels of urinary acid-stable inhibitors may prove to be useful markers of the disease.

Aged↗

PubMed1996

Dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn inhibits the proliferation rate of HL-60 cells.

Small acidic phosphorylated chromatin peptides show regulatory activity on gene expression. The peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn, synthesized on the basis of structural and biochemical studies, shows functional properties in vitro (phosphorylation by casein kinase II, control of DNA transcription by RNA polymerase II, inhibition of proliferation and promotion of differentiation in some cell lines) very similar to those of native chromatin peptides. In this report we show that the dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn remarkably inhibits cell growth of the HL-60 cell line. The biological effect of the peptide seems to be considerably higher than that shown by the nondansylated peptide, and it cannot be attributed to a toxic effect of the Dns group. The measurement of uptake of 3H-labelled Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn demonstrates that it is unable to pass through the HL-60 cell membrane. It is our considered opinion that the addition of hydrophobic groups to the peptide N-terminus should increase the biological activity by improving its transport through the cellular membrane.

Cell Division↗

PubMed1995

Synthesis and biological evaluation of N6-cycloalkyl derivatives of 1-deazaadenine nucleosides: a new class of anti-human immunodeficiency virus agents.

A series of 1-deazaadenine nucleosides with the N6 nitrogen unsubstituted or bearing methyl or cycloalkyl substituents, with or without a chloro group in the 2-position, and with the glycosylic moiety being ribose (1-16), 2'-deoxyribose (17-32), or 2', 3'-dideoxyribose (33-48) were designed and synthesized starting from 5,7-dichloro-3H-imidazo[4,5-b] pyridine (50). These compounds were evaluated for their in vitro activity against human immunodeficiency virus type-1 (HIV-1) and herpes simplex virus type-1 (HSV-1). In addition they were tested for their ability to inhibit adenosine deaminase (ADA) from calf intestine. While the parent compounds 1-deazaadenosine (9), 2'-deoxy-1-deazaadenosine (25), and 2',3'-dideoxy-1- deazaadenosine (41) and the corresponding 2-chloro derivatives were inactive, nucleosides bearing cycloalkyl substituents on N6 exhibited moderate to good anti-HIV-1 activity, compared to 2',3'-dideoxyadenosine, with the degree and pattern of improvement depending on the structure of the sugar moiety. In general, 2'-deoxy- and 2',3'-dideoxy derivatives were more potent compounds than the corresponding ribose nucleosides. Compounds bearing a 6-cycloheptyl or cyclooctylamine were the most active in every series. The presence of a chloro group in the 2-position improved both activity and therapeutic index in every series, the most active compound being 2'-deoxy-2-chloro-N6-cycloheptyl-1-deazaadenosine (23; ED50 = 0.2 microM). On the other hand, most of these derivatives were inactive as anti-HSV-1 agents, showing a high degree of virus selectivity. The 1-deazaadenine derivatives were not substrates of adenosine deaminase, and some of them proved to be good inhibitors of the enzyme. However, the ADA inhibitory activity does not account for the antiviral potency since increased lipophilicity and steric hindrance of substituents resulted in derivatives much less active than the parent compounds.

Adenosine↗