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G Ramponi

Publications and source records attributed to G Ramponi.

At least 73 records · Page 4Linked to original sources
PubMed1998

Studies on enzymatic activity and conformational stability of muscle acylphosphatase mutated at conserved lysine residues.

An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.

Acid Anhydride Hydrolases↗
PubMed1997

Mechanism of acylphosphatase inactivation by Woodward's reagent K.

The organ common-type (CT) isoenzyme of acylphosphatase is inactivated by Woodward's reagent K (WRK) (N-ethyl-5-phenylisoxazolium-3'-sulphonate) at pH6.0. The inactivation reaction follows apparent pseudo first-order kinetics. The dependence of the reciprocal of the pseudo first-order kinetic constant (kobs) on the reciprocal WRK concentration reveals saturation kinetics, suggesting that the WRK forms a reversible complex with the enzyme before causing inactivation. Competitive inhibitors, such as inorganic phosphate and ATP, protect the enzyme from WRK inactivation, suggesting that this reagent acts at or near to the enzyme active site. The reagent-enzyme adduct, which elicits a strong absorption band with lambdamax at 346 nm, was separated from unreacted enzyme by reverse phase HPLC and the modified protein was cleaved with endoproteinase Glu-C to produce fragments. The HPLC fractionation gave two reagent-labelled peptides (peak 1 and peak 2) that were analysed by ion-spray MS and sequenced. The former is VFFRKHTQAE (residues 20-29 of human CT acylphosphatase) and the latter IFGKVQGVFFRKHTQAE (residues 13-29). MS demonstrated that both peptides are WRK adducts. A fragment ion with m/z of 1171, which is present in the mass spectrum of peak 1, has been identified as a WRK adduct of the peptide fragment 20-26. The lambdamax at 346 nm of WRK adduct suggests that the modified residue is His-25. Five recombinant enzymes mutated in residues included in the 20-29 polypeptide stretch have been produced. Analysis of their reactivities with WRK demonstrates that His-25 is the molecular target of the reagent as its modification causes the inactivation of the enzyme. Since both His-25-->Gln and His-25-->Phe mutants maintain high catalytic activity, we suggest that the observed enzyme inactivation is caused by the reagent (covalently bound to His-25), which shields the active site.

Acid Anhydride Hydrolases↗
PubMed1997

The 5'-untranslated region of the human muscle acylphosphatase mRNA has an inhibitory effect on protein expression.

The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5'-untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem-loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10-fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5'-untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.

Acid Anhydride Hydrolases↗
PubMed1997

c-Src activates both STAT1 and STAT3 in PDGF-stimulated NIH3T3 cells.

Treatment of cells with PDGF and EGF specifically induces STAT1 and STAT3, which became phosphorylated on tyrosine residues to form homo and heterodimers: in these configurations they translocate into the nucleus where they act as transcription activators. However little is known about the activation of STATs in growth factor receptor signal transduction. Recently it has been shown that v-Src modulates the tyrosine phosphorylation of STAT3 but not of STAT1. Here we report that the cellular Src tyrosine kinase is involved in the activation of both STAT1 and STAT3 in PDGF stimulated NIH3T3 cells. Both tyrosine phosphorylation and DNA binding activity of STAT1 and STAT3 are up-regulated in c-Src overexpressing cells, while we observe the opposite phenomenon in cells overexpressing the dominant negative Src. Furthermore, our results show that STAT1 co-immunoprecipitates with c-Src, suggesting that the activation of STATs by Src occurs via a direct interaction. Taken together, these data suggest that c-Src is involved in activation of both STAT1 and 3 in PDGF signal transduction.

3T3 Cells↗
PubMed1997

Common-type acylphosphatase: steady-state kinetics and leaving-group dependence.

A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 degrees C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme-substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O-inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. all these findings, as well as the strong dependence of kcat upon the leaving group pK1, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.

Acid Anhydride Hydrolases↗
PubMed1997

LMW-PTP is a negative regulator of insulin-mediated mitotic and metabolic signalling.

To understand the physiological role of low Mr weight phosphotyrosine protein phosphatase (LMW-PTP) in insulin mediated signaling, we established clonal cell lines overexpressing the dominant negative (C12S mutant) LMW-PTP (dnLMW-PTP) from NIH3T3 murine fibroblasts expressing insulin receptor. Upon insulin stimulation we observe an association between the dnLMW-PTP and the beta-subunit of the insulin receptor. This association is dependent on the tyrosine phosphorylation of the insulin receptor since it is not observed in unstimulated cells. Furthermore, in vitro binding experiments between dnLMW-PTP and the insulin receptor reveal that the interaction is mediated by the LMW-PTP catalytic site, as indicated by competition with orthovanadate. DnLMW-PTP overexpression influences both the mitogenic and the metabolic bioeffects of insulin. In particular, in cells overexpressing dnLMW-PTP we observe an increase in the glycogenosynthesis rate and in mitosis as indicated by glucose incorporation into glycogen and thymidine incorporation into DNA, respectively. Moreover, we studied the insulin mediated signal transduction pathways starting from insulin receptor, such as the Src kinase, the p21Ras/ERK, and the PI3K routes. Our findings are consistent with a specific regulation of mitogenesis by LMW-PTP through a pathway involving c-Src kinase but independent by both PI3K and ERK. These data strongly suggest that LMW-PTP acts as a negative regulator of both mitogenetic and metabolic insulin signalling.

3T3 Cells↗
PubMed1997

Structure and function of the low Mr phosphotyrosine protein phosphatases.

Phosphotyrosine protein phosphatases (PTPases) catalyse the hydrolysis of phosphotyrosine residues in proteins and are hence implicated in the complex mechanism of the control of cell proliferation and differentiation. The low Mr PTPases are a group of soluble PTPases displaying a reduced molecular mass; in addition, a group of low molecular mass dual specificity (ds)PTPases which hydrolyse phosphotyrosine and phosphoserine/threonine residues in proteins are known. The enzymes belonging to the two groups are unrelated to each other and to other PTPase classes except for the presence of a CXXXXXRS/T sequence motif containing some of the catalytic residues (active site signature) and for the common catalytic mechanism, clearly indicating convergent evolution. The low Mr PTPases have a long evolutionary history since microbial (prokaryotic and eukaryotic) counterparts of both tyrosine-specific and dsPTPases have been described. Despite the relevant number of data reported on the structural and catalytic features of a number of low Mr PTPases, only limited information is presently available on the substrate specificity and the true biological roles of these enzymes, in prokaryotic, yeast and eukaryotic cells.

Amino Acid Sequence↗
PubMed1997

Evidence for glutathione involvement in platelet-derived growth-factor-mediated signal transduction.

Recent studies show that glutathione, while being involved in the well-known physiological processes of amino acid transport and detoxification, can also play a part in cell proliferation events. Cell treatment with l-buthionine sulphoximine, which causes glutathione depletion, is accompanied by a decrease in cell proliferation. At present no precise relationship between this thiol and any critical intermediate of the mitogenic cascade has been proved. In this study, conducted on NIH/3T3 murine fibroblasts, we demonstrate a strict correlation between glutathione levels and platelet-derived growth-factor-receptor activation in response to stimulation and cell proliferation. The receptor autophosphorylation is severely impaired at low glutathione cellular levels. The interaction of glutathione with this growth-factor receptor in vivo, while being rather specific, is complex and may involve both cytosolic and extracellular receptor domains.

3T3 Cells↗
PubMed1997

Structural and kinetic investigations on the 15-21 and 42-45 loops of muscle acylphosphatase: evidence for their involvement in enzyme catalysis and conformational stabilization.

The structural and catalytic importance of the 15-21 and 42-45 loop residues of the acylphosphatase muscular isoenzyme has been investigated by oligonucleotide-directed mutagenesis. Seven mutants involving conserved residues of the two loops have been prepared and characterized for structural, kinetic, and stability features by using different spectroscopic techniques and compared to the wild-type enzyme. The results are discussed in light of the crystal structure of the highly homologous common type acylphosphatase [Thunnissen et al. (1997) Structure 5, 69-79]. A differential role of the two loops has emerged: the 15-21 and the 42-45 loops appear mainly involved in active site formation and enzyme structural stabilization, respectively. These conclusions are supported by a strong impairment of the catalytic efficiency, in terms of enzymatic activity and substrate binding capability, for most of the 15-21 loop mutants. In particular, the Gly15Ala mutant is completely inactive and displays a native-like overall fold, indicating that the correct geometry of the 15-21 loop is an essential requisite for optimal enzymatic catalysis. Instead, the Gly45Ala mutant, though revealing unchanged catalytic properties, shows a considerably reduced conformational stability, as judged by circular dichroism and 1H NMR spectroscopy. This finding confirms previous results relative to Thr42 and Thr46 residues [Taddei et al. (1996) Biochemistry 35, 7077-7083] underlining the structural importance of the 42-45 loop as a linker for the two beta alpha beta units constituting the overall enzyme structure.

Acid Anhydride Hydrolases↗
PubMed1997

Differential migration of acylphosphatase isoenzymes from cytoplasm to nucleus during apoptotic cell death.

The subcellular distribution of both muscle and common type acylphosphatases was studied during apoptosis. Both isoenzymes were previously shown to have in vitro nucleolytic activity having a pH optimum at 6.8. In this paper we demonstrate a nuclear migration of the muscle type isoenzyme in response to various apoptotic stimuli either in K562 or in Jurkat cells, while the common type acylphosphatase isoform remains cytoplasmatic under the same conditions. Furthermore, we present evidences of a direct in vivo interaction between muscle acylphosphatase and two other DNAses of about 60 and 80 kDa in size. Our results are consistent with an in vivo involvement of the muscle isoform in apoptosis, possibly as a part of a multimeric protein complex that binds and hydrolyses DNA during this process.

Acid Anhydride Hydrolases↗
PubMed1997

Crystal structure of common type acylphosphatase from bovine testis.

BACKGROUND: Acylphosphatase (ACP) is a low molecular weight phosphomonohydrolase catalyzing with high specificity the hydrolysis of the carboxyl-phosphate bond present in acylphosphates. The enzyme is thought to regulate metabolic processes in which acylphosphates are involved, such as glycolysis and the production of ribonucleotides. Furthermore the enzyme is capable of hydrolyzing the phospho-aspartyl intermediate formed during the action of membrane pumps such as (Ca2++Mg2+) ATPase. Although the tertiary structure of a muscle ACP has been determined by NMR spectroscopy, little is known about the catalytic mechanism of ACP and further structures might provide an increased understanding. RESULTS: The structure of 'common type' ACP from bovine testis has been determined by X-ray crystallography to a resolution of 1.8 A. The structure has been refined to an R factor of 17.0 % using all data between 15 and 1.8 A. The binding of a sulphate and a chloride ion in the active centre allows a detailed description of this site. The overall protein folds of common type and muscle ACP are similar but their loops have very different conformations. These differences, in part, are probably caused by the binding of the ions in the active site of the common type form. The phosphate-binding loop of ACP shows some remarkable similarities to that of low molecular weight protein tyrosine phosphatase. CONCLUSIONS: The active site of ACP has been located, enabling a reaction mechanism to be suggested in which the phosphate moiety bound to Arg23 acts as a base, abstracting a proton from a nucleophilic water molecule liganded to Asn41. The transition-state intermediate is stabilized by the phosphate-binding loop. We suggest the catalysis to be substrate assisted, which probably explains why this enzyme can only hydrolyze acylphosphates.

Acid Anhydride Hydrolases↗
PubMed1997

Alteration of free calcium levels and acylphosphatase muscular isoenzyme in cultured dystrophic skin fibroblasts.

Levels of free intracellular calcium have been measured on two cell lines of cultured human fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. Both cell lines elicited a markedly higher content of the cation (98 nM and 57 nM, respectively) than control fibroblasts (35 nM). Differences toward controls were statistically significant (p < 0.01). Dystrophic fibroblasts were also found to possess a significantly reduced amount by about 50% of muscular acylphosphatase isoenzyme as compared to normal cells. As acylphosphatase was demonstrated to be involved in the regulation of Ca2+-ATPase activity from different sources, a hypothesis was formulated that could explain the disruption of calcium homeostasis as an effect of the altered acylphosphatase activity.

Acid Anhydride Hydrolases↗
PubMed1997

Insights into acylphosphatase structure and catalytic mechanism.

Acylphosphatase is one of the smallest enzymes known (about 98 amino acid residues). It is present in organs and tissues of vertebrate species as two isoenzymes sharing over 55% of sequence homology; these appear highly conserved in differing species. The two isoenzymes can be involved in a number of physiological processes, though their effective biological function is not still certain. The solution and crystal structures of different isoenzymes are known, revealing a close packed protein with a fold similar to that shown by other phosphate-binding proteins. The structural data, together with an extended site-directed mutagenesis investigation, led to the identification of the residues involved in enzyme catalysis. However, it appears unlikely that these residues are able to perform the full catalytic cycle: a substrate-assisted catalytic mechanism has therefore been proposed, in which the phosphate moiety of the substrate could act as a nucleophile activating the catalytic water molecule.

Acid Anhydride Hydrolases↗
PubMed1997

Structural, catalytic, and functional properties of low M(r), phosphotyrosine protein phosphatases. Evidence of a long evolutionary history.

The PTPase family comprises a number of classes of functionally and structurally unrelated enzymes; it represents an important component of the protein-tyrosine phosphorylation/dephosphorylation machinery, which regulates the level of tyrosine phosphorylation of a number of intracellular proteins. A wealth of recently reported data indicates growing interest in a group of PTPases characterized by low (near 20 kDa) molecular weight and high sequence homology (low M(r), PTPases). These enzymes are present in organisms spanning the philogenetic scale, from prokaryotes to yeast and mammals. The sequence homology of the low M(r), PTPases with other classes of PTPases is limited to the active site sequence CXXXXXRS/T, containing the Cys and Arg residues involved in enzyme catalysis found in all PTPases. The X-ray structural data of three enzymes belonging to different classes of PTPases, a bovine liver low M(r), PTPase isoenzyme, PTP1B, and Yersinia PTPase, show that all these enzymes maintain the same active site and overall catalytic mechanism, though displaying different chain foldings and topologies, supporting convergent evolution. Limited findings on the in vivo function of the low M(r), PTPases are presently available; however, an involvement of the mammalian enzymes in the membrane growth factor receptor signal transduction is emerging. The distribution of these enzymes in philogenetically distant unicellular and multicellular organisms supports their participation in important cell functions.

Amino Acid Sequence↗
PubMed1997

Acylphosphatase is involved in differentiation of K562 cells.

The level of both isoforms of acylphosphatase was evaluated in the human erythroleukemia K562 cell line during differentiation. K562 cells were treated with PMA, which induces megakaryocytic differentiation, and with aphidicolin or hemin, which stimulate erythrocytic differentiation. While the MT isoform showed an average 10-fold increase independently of the differentiating agent used, only hemin treatment caused a similar increase of the CT isoform, suggesting a different role of the two isoforms in the cell. Treatment with either hemin or aphidicolin of K562 cells overexpressing the two acylphosphatase isoforms suggested the possibility that acylphosphatases play a role in the onset of differentiation.

Journal Article↗
PubMed1997

Alteration of intracellular free calcium and acylphosphatase levels in differentiating SH-SY5Y neuroblastoma cells.

Levels of acylphosphatase isoenzymes and free intracellular calcium have been investigated in cultured SH-SY5Y human neuroblastoma cells under stimulation with all-trans retinoic acid and phorbol-12-myristate-13-acetate. Under these conditions morphological and functional characteristics demonstrated the differentiation of SH-SY5Y cells towards neuronal phenotype. Retinoic acid treatment caused a progressive and synchronous increase of the organ common-type acylphosphatase and of free intracellular calcium but not of the muscle-type acylphosphatase. Phorbol-12-myristate-13-acetate treatment gave rise to a peak of the muscle-type acylphosphatase levels during the early differentiation stage whereas organ common-type isoenzyme and free calcium levels show a pattern similar to that observed in retinoic acid-treated cells. These evidences indicate that the two acylphosphatase isoenzymes play different roles in SH-SY5Y differentiation and that during this process the expression of organ common-type acylphosphatase increases in a synchronous way with intracellular free calcium concentration.

Acid Anhydride Hydrolases↗
PubMed1996

Identity of zinc ion-dependent acid phosphatase from bovine brain and myo-inositol 1-phosphatase.

A 62 kDa Zn(2+)-dependent acid phosphatase has been purified from bovine brain. The protein was carboxymethylated and then cleaved by endoproteinase Glu-C, trypsin and CNBr. Several fragments were subjected to structural analysis either by using mass spectrometry or automated peptide sequencing. The four sequenced peptides were compared with the known protein sequences contained in the EMBL Data Bank. All four peptide sequences were identical to the corresponding amino-acid sequences present in myo-inositol 1-phosphatase from bovine brain. Furthermore we found that the amino-acid composition of Zn(2+)-dependent acid phosphatase purified in our laboratory is very similar to that of myo-inositol 1-phosphatase, and that several peptide fragments have molecular weights (measured by mass spectrometry techniques) identical to those expected for cleavage-fragments originated from the authentic myo-inositol 1-phosphatase. This is one of the key enzymes in the receptor-stimulated inositol phospholipid metabolism and it has been considered as the probable target of Li+ ion during LiCl therapy in manic-depressive patients. The comparison of the Zn(2+)-dependent acid phosphatase and the Mg(2+)-dependent myo-inositol-1-phosphatase activities, measured at different purification steps, shows that the ratio between the two activities was remarkably constant during enzyme purification. We also demonstrated that in the presence of Mg2+ this enzyme efficiently catalyses the hydrolysis of myo-inositol 1-phosphate, and that the Li+ ion inhibits this activity. Furthermore, the thermal treatment of the enzyme causes a time-dependent parallel decrease of both Zn-dependent p-nitrophenyl phosphatase (assayed at pH 5.5) and Mg(2+)-dependent myo-inositol-1-phosphatase (assayed at pH 8.0) activities, suggesting the hypothesis that the same protein possesses both these activities.

Acid Phosphatase↗