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161.
Giovanna Cacciapuoti Maria Angela Moretti Sabrina Forte Assunta Brio Laura Camardella Vincenzo Zappia Marina Porcelli 《European journal of biochemistry》2004,271(23-24):4834-4844
The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins. 相似文献
162.
Vertebrate fossils from many different formations fluoresce when exposed to ultraviolet (UV) light. In this paper field observations and controlled experiments in the Chadron Formation (White River Group, late Eocene) of Wyoming are used to assess the utility of searching for fossils at night using ultraviolet light. The results indicate that, especially for very small teeth and egg-shell fragments, searching with ultraviolet light at night can result in significantly more specimens than searching during daylight hours. This method has the potential to increase sample sizes for small vertebrate specimens that are often overlooked when using standard collecting techniques. 相似文献
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164.
Beatriz M. Brena Claudia Pazos Laura Franco-Fraguas Francisco Batista-Viera 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,684(1-2)
This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases. 相似文献
165.
Daniela Pinzani Anna Maria Papini Mario Chelli Mauro Ginanneschi Carlo Alberto Maggi Riccardo Patacchini Laura Quartara Gianfranco Rapi 《Letters in Peptide Science》1996,2(5):307-313
Summary The adamantane moiety was introduced in the tachykinin NK2 receptor-selective agonist [-Ala8]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH2, MEN 10210) and in different positions of the NK2 receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2) in order to investigate how this substitution affects their biological activity at tachykinin NK1, NK2 and NK3 receptors. 1-Adamantaneacetic acid (1-Ada-CH2COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH2CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH2). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH2CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH2CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH2CO)-NH2]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK1 and NK2 receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK3 receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK1, NK2 and NK3 receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK2 receptors of the rabbit isolated pulmonay artery. 相似文献
166.
167.
Vittorio Moretto Fernando Formaggio Marco Crisma Gian Maria Bonora Claudio Toniolo Ettore Benedetti Antonello Santini Michele Saviano Benedetto Di Blasio Carlo Pedone 《Journal of peptide science》1996,2(1):14-27
A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic Cα,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac8c), and two Ala/Ac8c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and 1H-NMR. The molecular structures of the amino acid derivative Z-Ac8c-OH, the dipeptide pBrBz- (Ac8c)2-OH and the tripeptide pBrBz-(Ac8c)3-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac8c-NHMe. Taken together, the results obtained strongly support the view that the Ac8c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of Cα, α-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n=3, 5–7) investigated so far. The implications for the use of the Ac8c residue in peptide conformational design are considered. 相似文献
168.
169.
170.
Carbonera Daniela; Rovati Laura; Guano Fulvio; Balestrazzi Alma 《Journal of experimental botany》1995,46(3):347-354
Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg2+ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC50 32 mmol m3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation 相似文献