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Query: UMLS:C0272170 (SDS)
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Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohydrate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d-glucosamine, d-galactosamine and d-mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.
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PMID:Characterization of the lectin from females of Phlebotomus duboscqi sand flies. 1247 26

Mass spectrometry was used to identify the sites of covalent attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the covalent attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative covalent attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The covalent attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.
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PMID:Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol covalent attachment sites on estrogen receptor alpha. 1250 Dec

A new class of poly(ADP-ribose) (pADPr)-binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), has been identified by a proteomic approach using matrix-assisted laser-desorption-ionization time-of-flight ('MALDI-TOF') MS. Liquid-phase isoelectric focusing with a Rotofor cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotofor protein fractions were further separated by SDS/PAGE and then transferred to a PVDF membrane. pADPr-binding proteins were analysed by autoradiography of the protein blot after incubation with (32)P-labelled automodified pADPr polymerase-1 (PARP-1). Peptide mass fingerprinting of selected bands identified the most abundant pADPr-binding proteins as hnRNPs, a family of proteins that bind pre-mRNA into functional complexes involved in mRNA maturation and transport to the cytoplasm. Sequence homology database searching against a previously reported pADPr-binding sequence motif revealed that the hnRNPs contain a putative pADPr-binding sequence pattern [Pleschke, Kleczkowska, Strohm and Althaus (2000) J. Biol. Chem. 275, 40974-40980]. pADPr-binding assays performed with synthetic peptides by the dot-blot technique and with nitrocellulose-transferred recombinant hnRNPs confirmed the pADPr-binding protein identification and the specificity of the interaction. These results could establish a link between increased levels of pADPr in DNA damaged cells and the modified protein expression pattern resulting from altered mRNA trafficking.
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PMID:A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins. 1251 4

Three antibacterial peptides, named protaetins 1, 2, and 3, were purified and characterized from immunized larval hemolymph of Protaetia brevitarsis, a fruit tree pest in Korea. Also, protaetin 1 was cloned. Acid extraction, gel filtration, preparative acid-urea PAGE, and reversed-phase FPLC were used for purification of peptides. Protaetins 1 and 3 had molecular masses of 7.5 and 12 kDa on Tricine SDS-PAGE, respectively, and the molecular mass of protaetin 2 was 9,283.95 Da as determined by MALDI-TOF mass spectrometry. In an antibacterial assay, protaetins showed antibacterial activities against a panel of Gram-positive and -negative bacteria. For the RT-PCR (reverse transcription polymerase chain reaction) to obtain the complete primary sequence, the primer was designed according to the N-terminal amino acid sequence of protaetin 1. Amino acid sequence homology of protaetin 1 with holotricin 2, an antibacterial peptide from Holotrichia diomphalia, showed 99% identity. Northern blot analysis showed that the protaetin 1 gene was strongly expressed in the fat body after Escherichia coli injection, but not in normal fat body. Also, it was expressed in the gut, but was much weaker after immunization.
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PMID:Purification and cDNA cloning of inducible antibacterial peptides from Protaetia brevitarsis (Coleoptera). 1252 64

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.
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PMID:Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers. 1253 6

Nodamura virus (NoV) was the first isolated member of the Nodaviridae, and is the type species of the alphanodavirus genus. The alphanodaviruses infect insects; NoV is unique in that it can also lethally infect mammals. Nodaviruses have bipartite positive-sense RNA genomes in which RNA1 encodes the RNA-dependent RNA polymerase and the smaller genome segment, RNA2, encodes the capsid protein precursor. To facilitate the study of NoV, we generated infectious cDNA clones of its two genomic RNAs. Transcription of these NoV1 and NoV2 cDNAs in mammalian cells led to viral RNA replication, protein synthesis, and production of infectious virus. Subgenomic RNA3 was produced during RNA replication and encodes nonstructural proteins B1 and B2 in overlapping ORFs. Site-directed mutagenesis of these ORFs, followed by SDS-PAGE and MALDI-TOF mass spectrometry analyses, showed synthesis of B1 and two forms of B2 (B2-134 and B2-137) during viral replication. We also characterized a point mutation in RNA1 far upstream of the RNA3 region that resulted in decreased RNA3 synthesis and RNA2 replication, and a reduced yield of infectious particles. The ability to reproduce the entire life cycle of this unusual nodavirus from cDNA clones will facilitate further analysis of NoV RNA replication and pathogenesis.
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PMID:Recovery of infectivity from cDNA clones of nodamura virus and identification of small nonstructural proteins. 1257 89

This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.
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PMID:Phenol hydroxylase from Acinetobacter radioresistens S13. Isolation and characterization of the regulatory component. 1265 98

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are receptors for several Neisseria and Haemophilus spp. In this investigation, we demonstrate that a major outer membrane protein of Moraxella catarrhalis (Mx) strains, belonging to the ubiquitous surface protein (Usp) family, also interacts with the receptor. The interaction was demonstrated in Western blot overlay of SDS-PAGE-separated bacterial proteins using soluble receptor constructs as well as by co-precipitation experiments. The identity of the bacterial ligand was further ascertained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). It was shown to belong to the UspA1 subfamily. In general, antibodies raised against synthetic UspA1, but not UspA2, peptides bound to the Mx ligand. CEACAM1-Fc-binding property could be demonstrated in all the clinical isolates examined but varied between strains. A single colony derivative of an Mx isolate was also demonstrated to bind to transfected Chinese hamster ovary and some human respiratory epithelial cells in a CEACAM-dependent manner. Thus, we have identified the third respiratory pathogen with the capacity to target the CEACAM family of receptors. The Mx ligand is structurally unrelated to those of Neisseria and Haemophilus.
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PMID:A novel cell-binding mechanism of Moraxella catarrhalis ubiquitous surface protein UspA: specific targeting of the N-domain of carcinoembryonic antigen-related cell adhesion molecules by UspA1. 1265 49

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.
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PMID:Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging. 1265 91

Two extracellular isoenzymes of polygalacturonases PG1 and PG2 were isolated from 3-day-old culture filtrates of Trichoderma reesei. The two enzymes were purified to homogeneity by ion-exchange, gel filtration and hydrophobic interaction chromatographies. PG1 and PG2 exhibit similar molecular weights from gel filtration and SDS-PAGE. Their properties, including optimal pH and temperature, thermal stability and Km were compared. Characterization of substrate specificity showed that the two enzymes had higher affinity toward PGA (B0100) derived from sugar beet pectin (SBP) than PGA from lime pectin. A series of SBPs with different distribution patterns of methyl and acetyl groups, produced by treatment with either plant pectin methylesterase (P-series) or fungal pectin methylesterase (F-series) or base catalysis (B-series), was used as substrates for PG1 and PG2. Substrates with a low degree of esterification were preferred substrates. The activities of PG1 and PG2 were strongly correlated to the degree of methylation and very little effect from acetylation. The products generated by digestion of selected lime and SBPs were analysed using matrix assisted laser desorption ionisation time of flight (MALDI TOF) MS. A mode of action revealed a random cleavage pattern for PG1 and PG2, confirming that these enzymes are endopolygalacturonases.
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PMID:New polygalacturonases from Trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins. 1266 7


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