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M Porcelli

Publications and source records attributed to M Porcelli.

At least 19 recordsLinked to original sources
PubMed2001

Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus.

The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-A resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 A. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr(89), which corresponds to serine in E. coli PNP, and Glu(163), which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr(89) is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu(163). This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120 degrees C and retains full activity after 2 h at 100 degrees C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.

Binding Sites↗
PubMed2001

Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus.

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.

Binding Sites↗
PubMed2001

X-ray photoelectron spectra of Pd(II) and Pt(II) complexes with 1,3-thiazolidine-2-thione. A quantum mechanics study on the free ligand.

Metal complexes of general formula M(ttz)2X2 (with M= Pd(II) or Pt(II); X = Cl or Br; ttz = 1,3-thiazolidine-2-thione) have been synthetized as crystalline compounds and studied by means of X-ray Photoelectron Spectroscopy (XPS). The chemical shift of core level signals showed that ttz is bonded to the metal through the thioketonic sulphur atom and that electronic charge redistribution in the ligand takes place after complexation. No metal-nitrogen bonds are present. This is consistent with the results of all the quantum mechanical models according to which hydrogen is bound to nitrogen, even in the hydrogen bonded complex, making the latter rather unavailable to coordination

Ligands↗
PubMed2001

Cyclic vomiting syndrome: in vitro nitric oxide and interleukin-6 release by esophageal and gastric mucosa.

Cyclic vomiting syndrome is a disorder characterized by recurrent episodes of nausea and vomiting with complete resolution of symptoms between attacks. Nitric oxide plays a critical role in regulating several components of gastrointestinal mucosal defense and injury. Interleukin-6 has a wide variety of actions in the gastrointestinal apparatus. The purpose of this study was to evaluate the synthesis and release of nitric oxide and interleukin-6 by the esophageal and gastric mucosa in 10 children with cyclic vomiting syndrome, during symptom-free periods, and in 10 controls. The nitric oxide and interleukin-6 release by esophageal mucosa cells obtained from cyclic vomiting patients was quite similar to that in controls, but the release of nitric oxide from gastric mucosa cells of patients was significantly higher than that of controls. Conversely, no interleukin-6 was detectable in gastric mucosa cell supernatants in any of the patients. Further studies are needed to evaluate the relationship between factors triggering cyclic vomiting syndrome and the release of nitric oxide and interleukin-6 by gastric mucosa.

Cells, Cultured↗
PubMed2000

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus.

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.

Adenosylhomocysteinase↗
PubMed2000

Sandifer's syndrome in a breast-fed infant.

Sandifer's syndrome is a rare manifestation of gastroesophageal reflux (GER) in children, occurring in association with abnormal movements of the head, neck, and upper part of the trunk. Out of 65 children with Sandifer's syndrome described in literature, only 2 were breast-fed. We report on a 15-day-old breast-fed girl affected by Sandifer's syndrome. Pathological GER was diagnosed with 24 h pH esophageal monitoring. In our patient, all the symptoms of Sandifer's syndrome disappeared when she was cow's milk formula-fed. The role of food allergy to dietary proteins ingested by a lactating mother is discussed.

Breast Feeding↗
PubMed1999

Cyclic pentapeptides of chiral sequence DLDDL as scaffold for antagonism of G-protein coupled receptors: synthesis, activity and conformational analysis by NMR and molecular dynamics of ITF 1565 a substance P inhibitor.

Under the hypotheses of a structurally related binding site for antagonists of G-protein coupled receptors and the ability of cyclic pentapeptides of chiral sequence D1L2D3D4L5 to form rigid structures with which probe the pharmacophoric specificity of these receptors, inhibitors of substance P were designed based on available structure-activity relationships. ITF 1565, cyclo[D-Trp1-Pro2-D-Lys3-D-Trp4-Phe5], antagonized substance P activity mediated by type 1 neurokinin receptor (NK1) whereas it acted weakly against NK2 and did not inhibit endothelin at all. The preferential conformation of the peptide was obtained from nmr spectroscopy and computer calculations, and shown to contain the same beta II-turn and gamma'-turn found in other cyclic pentapeptides with the same chiral sequence. The structure of the peptide was compared with that of the beta-D-glucose molecule that has been proposed as a semirigid scaffold for antagonists of G-protein coupled receptors. The gamma'-turn of the cyclic peptide superimposed well with beta-D-glucose in the chair conformation. Furthermore, when the side chains were considered, the aromatic groups of the two molecules were found to generally overlap. These results support the view of G-protein coupled receptors as possessing structurally similar binding sites for antagonists and suggest that cyclic pentapeptides of chiral sequence D1L2D3D4L5 may be useful as semirigid scaffolds for the design of antagonists of this family of receptors.

Animals↗
PubMed1999

Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.

The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the original S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds. On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S. solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding.

Base Sequence↗
PubMed1997

Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system.

Two thermophilic and thermostable enzymes, isolated from Sulfolobus solfataricus, S-adenosylhomocysteine hydrolase and 5'-methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non-thermal microwave effects. The exposure causes a non-thermal, irreversible and time-dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of S-adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

Adenosylhomocysteinase↗
PubMed1996

Cloning and sequencing of the gene coding for S-adenosylhomocysteine hydrolase in the thermophilic archaeon Sulfolobus solfataricus.

The gene from the thermophilic archaeon Sulfolobus solfataricus (Ss), encoding the S-adenosylhomocysteine hydrolase (AdoHcyHD), has been cloned. Two degenerate oligodeoxyribonucleotide (oligo) probes, synthesized on the basis of amino acid (aa) sequence of cyanogen bromide-peptide fragments of the purified protein, were used to screen a genomic library of Ss cloned into the pGEM7Zf(+) vector. The AdoHcyHD gene (adohcyhd) comprises 1254 nucleotides (nt) and encodes a polypeptide of 417 aa with a deduced molecular mass of 46 kDa, in good agreement with the value directly measured for the purified enzyme. The identity of more than 32% of the deduced aa sequence was confirmed by Edman degradation of peptides. Putative regulatory elements which are in good agreement with the archaeal promoter consensus sequences were identified in the flanking regions. Comparison of the aa sequences of AdoHcyHD from different sources shows a remarkable degree of conservation. Surprisingly, several aa residues, thought important in substrate binding and catalysis, show non-conserved replacements in Ss AdoHcyHD.

Adenosylhomocysteinase↗
PubMed1996

Extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Gene cloning and amino acid sequence determination.

A gene encoding an extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase was cloned from the archaeon Sulfolobus solfataricus. Two degenerate oligodeoxyribonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the protein were used to screen a genomic library of S. solfataricus cloned into the pGEM7Zf(+) vector. The DNA fragment of 2118 bp containing the 5'-methylthioadenosine phosphorylase gene was sequenced. The open reading frame comprises 711 nucleotides, which includes the stop codon, and encodes a protein of 236 residues whose molecular mass is in good agreement with the value determined by gel filtration for the purified enzyme. The N- and C-terminal sequences of the protein and the sequences of the peptides prepared by cyanogen bromide cleavage exactly match with the corresponding sequences deduced from the gene, thus confirming the identity of the 5'-methylthioadenosine phosphorylase gene. Typical archaebacterial regulatory sites were identified in the flanking regions and a potential Shine-Dalgarno-like sequence was recognized around the ATG initiation codon. The deduced amino acid sequence showed 32% identity and 30% identity with Escherichia coli purine-nucleoside phosphorylase and with E, coli uridine phosphorylase, respectively. Evolutionary and structural implications of this similarity are discussed.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds.

5'-Methylthioadenosine phosphorylase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity. Reducing agents are not required for catalytic activity. The enzyme has a molecular mass of 160 kDa and is composed of six apparently identical subunits of 27 kDa. The NH2-terminal sequence shows high homology (50%) with the NH2-terminal sequence of Escherichia coli purine nucleoside phosphorylase. Physicochemical and kinetic features are reported. 5'-Methylthioadenosine phosphorylase is highly thermophilic, with an optimum temperature of 120 degrees C. The enzyme is characterized by extreme thermal stability, remaining completely active after 2 h at 100 degrees C and showing half-inactivation times of 15 and 5 min when incubated at 130 and 140 degrees C, respectively. An apparent melting temperature of 132 degrees C has been calculated. After 24 h of incubation at room temperature no loss of activity is detected in the presence of 9 M urea, 4 M guanidine hydrochloride, 0.075% SDS, 50% methanol, 50% ethanol, 50% dimethylformamide, 1 M NaCl, and 1% Triton X-100. Data are also reported on the enzyme's resistance to proteolysis and on the effect of salts, detergents, solvents, and reducing agents on enzyme thermostability. Labeling experiments with iodo[2-14C]acetic acid resulted in the incorporation of approximately 12 mol of labeled iodoacetate/mol of protein, indicating the presence of six disulfide bonds that, on the basis of SDS-polyacrylamide gel electrophoresis, are probably positioned intersubunits, resulting in the organization of the enzyme into two trimers. 5'-Methylthioadenosine (MTA) phosphorylase is endowed with a broad substrate specificity, being able to phosphorolytically cleave inosine, guanosine, and adenosine with a better efficiency than MTA, allowing us to hypothesize that in S. solfataricus the same enzyme is responsible for the catabolism of MTA and of these purine nucleosides.

Amino Acid Sequence↗
PubMed1993

S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus: purification, physico-chemical and immunological properties.

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 161 nmol of S-adenosylhomocysteine formed per min per mg of protein, and the overall yield, by immunoaffinity purification, is 51%. The enzyme has a molecular mass of 190 kDa, is composed of four identical subunits (subunit mass 47 kDa), and contains four molecules of tightly-bound NAD+ per tetramer of which about 40% is in the reduced form. Physico-chemical features, including amino-acid composition and secondary structure, are reported. The pure protein, used to raise specific rabbit antisera, shows immunological properties different from other S-adenosylhomocysteine-metabolizing enzymes. The enzyme is thermophilic with an optimum temperature of 75 degrees C, and shows an apparent melting temperature of 95 degrees C by measuring its residual activity after 10 min incubation at increasing temperatures.

Adenosylhomocysteinase↗
PubMed1992

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium. 1. Conformational behavior of the oligomeric enzyme in solution.

The effect of temperature on the molecular structure of propylamine transferase from Sulfolobus solfataricus has been investigated. Sulfolobus solfataricus is an extreme thermophilic archaebacterium with an optimum living condition at 90 degrees C. The enzyme is an oligomeric (trimer) protein of molecular mass 112 kDa. The frictional ratio for the native protein suggests an irregularly shaped compact globular structure. The protein matrix is well organized as suggested by far ultraviolet circular dichroism at 25 degrees C (18% alpha helix, 43% beta structure, 19% beta bends and 20% unordered: root mean square = 7). Structural effects of temperature were investigated over 25-85 degrees C. The protein retains its quaternary structure in this temperature range. A highly reversible subtle conformational transition was detected by numerous structure-dependent techniques over 40-50 degrees C, with a midpoint centered at 45 degrees C. Functional data also support this view. In fact, two enzyme forms, characterized by different catalytic properties, are present in solution. The Arrhenius plot suggests the occurrence of two different activation-energy-dependent processes, one at a temperature higher and one at a temperature lower than 45 degrees C. The transition has been considered as a molecular switch between two protein populations at equilibrium with different functional and structural properties, temperature modulated. A physiological role for the molecular switch has also been postulated. The protein also shows some subtle and reversible spectroscopic changes around 75 degrees C. The molecular basis of the thermophilic nature of this enzyme seems to reside in its capability to dynamically couple catalytic and structural events to the thermal properties of the ambient medium.

Amino Acids↗
PubMed1992

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium. 2. Denaturation and structural stability.

The thermal stability of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium, has been characterized thermodynamically by a Van't Hoff analysis. Conformational transitions induced by guanidine hydrochloride, as well as by temperature, have been linked together in a scheme involving six equilibria, which arise from both dissociation and unfolding. The mechanism by which the protein achieves thermal stabilization is quite unusual. It is driven by a conformational equilibrium between two forms of different stability. The stability of each form towards denaturation is characterized by a specific temperature dependence. The low-temperature form, indicated as 'form A', is stable over 12-89 degrees C. Its stability maximum is 36.8 kJ/mol at 50 degrees C. 'Form B', which is populated at higher temperature, spans the interval 28-146 degrees C. Its stability maximum is 71.6 kJ/mol at 87 degrees C. A possible explanation for the mechanism underlying this behaviour is discussed assuming that two major terms contribute to stability, i.e. hydrophobic interactions arising from burying of the accessible surface residues as well as conformational entropy. The thermal stabilization of the enzyme seems to depend on effects related to both an overall increase of flexibility and a concomitant decrease of the area buried upon folding. In this regard proteins from extreme thermophilic organisms appear to be a useful model to shed new light on the general problem of protein stability.

Enzyme Stability↗
PubMed1991

S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.

S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein. S-Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S-adenosyl[Me-3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.

Adenosylmethionine Decarboxylase↗