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Gel Filtration (gel + filtration)
Terms modified by Gel Filtration Selected AbstractsORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activityFEBS JOURNAL, Issue 8 2002Nadia J. Kershaw The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ,,15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to >,95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate , and , subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an ,2,2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the , subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the , subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major , subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N -acetylornithine to glutamate, but not the formation of N -acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N -acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the , subunit. [source] A Lipoprotein-derived Antimicrobial Factor from Hen-egg Yolk is Active Against Streptococcus SpeciesJOURNAL OF FOOD SCIENCE, Issue 8 2002D. Brady ABSTRACT: Oral administration of hen-egg yolk provides protection against specific pathogens. We examined the antibacterial activity of fractionated egg yolk against 2 pathogenic Streptococcus strains, using an in vitro assay. A water-soluble protein fraction (WSPF) of egg yolk consistently inhibited the growth of S. mutans by 25%. The WSPF treated with pancreatin demonstrated > 80% inhibition of bacterial growth. Growth of S. sanguis was completely inhibited. Gel filtration and ion exchange chromatography established that anti-Streptococcal activity resided with lipoproteins. Antibacterial activity was released by crude lipase or a combination of lipase and protease treatment of egg lipoproteins. Thus, hen-egg yolk lipoproteins are important molecules for lipid-mediated antimicrobial activity. [source] Self-association of EPEC intimin mediated by the ,-barrel-containing anchor domain: a role in clustering of the Tir receptorMOLECULAR MICROBIOLOGY, Issue 1 2004Thierry Touzé Summary Outer membrane intimin directs attachment of enteropathogenic Escherichia coli (EPEC) via its Tir receptor in mammalian target cell membranes. Phosphorylation of Tir triggers local actin polymerization and the formation of ,pedestal-like' pseudopods. We demonstrate that the intimin protein contains three domains, a flexible N-terminus (residues 40,188), a central membrane-integrated ,-barrel (189,549), and a tightly folded Tir-binding domain (550,939). Intimin was shown by electron microscopy to form ring-like structures with a ,7 nm external diameter and an electron dense core, and to form channels of 50picoSiemens conductance in planar lipid bilayers. Gel filtration, multiangle light scattering and cross-linking showed that this central ,-barrel membrane-anchoring domain directs intimin dimerization. Isothermal titration calorimetry revealed a high affinity, single-binding site interaction of 2 : 1 stoichiometry between dimeric intimin and Tir, and modelling suggests that this interaction determines a reticular array-like superstructure underlying receptor clustering. In support of this model, actin rearrangement induced in Tir-primed cultured cells by intimin-containing proteoliposomes was dependent on the concentration of both intimin and Tir, and co-localized with clustered phosphorylated Tir. [source] Different proportions of cadmium occur as Cd-binding phytochelatin complexes in plantsPHYSIOLOGIA PLANTARUM, Issue 2 2007Eduardo Marentes The aim was to determine cadmium (Cd) speciation in various plants, between buffer-soluble and acid-soluble Cd, and also within the buffer-soluble Cd. A better understanding of Cd speciation shows the relative importance of different biological mechanisms for Cd sequestration. Roots of Pistia stratiodes, Eichhornia crassipes, Agrostis gigantea, Deschampsia caespitosa and wheat Triticum turgidum var. durum were analyzed. Buffer extractions solubilized varying proportions of Cd, ranging from 12% in Eichhornia to 83% in Agrostis. The proportion increased with time of Cd exposure in Pistia. It also increased in wheat roots as the external Cd rose from 0.05 to 0.5 ,M and was lowest in old leaves and highest in roots. The remaining Cd was extractable with acid. Gel filtration resolved buffer-soluble Cd into three peaks distinct from inorganic Cd. Two complexes with phytochelatins and related polythiols were present in all cases, inorganic Cd being prominent only in Eichhornia extracts. The phytochelatin complexes accounted for 2% of the root Cd in Eichhornia to 78% in Agrostis. In wheat, phytochelatins bound 82% of the Cd in roots, 19% in young leaves and 12% in old leaves. The cysteine-rich protein metallothionein from wheat was detected immunologically in the void volume of gel filtrations of old and young leaves, but not of roots, and was distinct from the two phytochelatin-based complexes. Speciation of Cd in the various plants indicated that phytochelatins were not necessarily the major ligands of Cd. [source] Characterization of heterotrimeric G protein complexes in rice plasma membraneTHE PLANT JOURNAL, Issue 2 2004Chiyuki Kato Summary Two genes in the rice genome were identified as those encoding the , subunits, ,1 and ,2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the ,, ,, ,1, and ,2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the , subunits were present in large protein complexes (about 400 kDa) containing the other subunits, ,, ,1, and ,2, and probably also some other proteins, whereas large amounts of the , and , (,1 and ,2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the , subunit interacted tightly with the ,1 and ,2 subunits, and so the , and , subunits appeared to form dimers in rice cells. Some dimers were associated with the , subunit, because few ,, ,1, and ,2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the , subunit. When a constitutively active form of the , subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form. [source] A subclass of myosin XI is associated with mitochondria, plastids, and the molecular chaperone subunit TCP-1, in maizeCYTOSKELETON, Issue 4 2004Zhengyuan Wang Abstract The role and regulation of specific plant myosins in cyclosis is not well understood. In the present report, an affinity-purified antibody generated against a conserved tail region of some class XI plant myosin isoforms was used for biochemical and immunofluorescence studies of Zea mays. Myosin XI co-localized with plastids and mitochondria but not with nuclei, the Golgi apparatus, endoplasmic reticulum, or peroxisomes. This suggests that myosin XI is involved in the motility of specific organelles. Myosin XI was more than 50% co-localized with tailless complex polypeptide-1, (TCP-1,) in tissue sections of mature tissues located more than 1.0 mm from the apex, and the two proteins co-eluted from gel filtration and ion exchange columns. On Western blots, TCP-1, isoforms showed a developmental shift from the youngest 5.0 mm of the root to more mature regions that were more than 10.0 mm from the apex. This developmental shift coincided with a higher percentage of myosin XI /TCP-1, co-localization, and faster degradation of myosin XI by serine protease. Our results suggest that class XI plant myosin requires TCP-1, for regulating folding or providing protection against denaturation. Cell Motil. Cytoskeleton 57:218,232, 2004. © 2004 Wiley-Liss, Inc. [source] Computer-assisted 2-D agarose electrophoresis of Haemophilus influenzae type B meningitis vaccines and analysis of polydisperse particle populations in the size range of viruses: A reviewELECTROPHORESIS, Issue 4 2007Dietmar Tietz Dr. Abstract When protein,polysaccharide conjugated vaccines were first developed for the immunization of small children against meningitis caused by infection with Haemophilus influenzae type b (Hib), the vaccine preparations varied in immunogenicity. Testing for immunogenicity was time-consuming and alternative analytical procedures for determining vaccine quality were unsatisfactory. For example, due to the very high molecular weight of the vaccine particles, immunogens could only be physically characterized as a fraction in the void volume of Sepharose gel filtration. In search of better analytical methods, a computer-assisted electrophoretic technique for analyzing such vaccines was developed in the period from 1983 to 1995. This new approach made it possible to analyze highly negatively charged particles as large as or larger than intact viruses. 2-D gel patterns were generated that varied depending on the conditions of the particular vaccine preparation and were therefore characteristic of each vaccine sample. Thus, vaccine particle populations with a continuous size variation over a wide range (polydisperse) could be characterized according to size and free mobility (related to particle surface net charge density). These advances are reviewed in this article, since the developed methods are still a promising tool for vaccine quality control and for predicting immunogen effectiveness in the production of vaccines. The technique is potentially beneficial for Hib immunogens and other high-molecular-mass vaccines. Additional biomedical applications for this nondenaturing electrophoretic technique are briefly discussed and detailed information about computational and mathematical procedures and theoretical aspects is provided in the Appendices. [source] Characterization of alanyl aminopeptidase from insecticide resistant and susceptible strains of Musca domestica L.ENTOMOLOGICAL RESEARCH, Issue 3 2008Sohail AHMED Abstract To investigate the high activity of intracellular proteases in insecticide resistant strains of Musca domestica L., purification by anion-exchange chromatography and gel filtration of one of the enzymes, alanyl aminopeptidase (Ala AP), in three strains of Musca domestica was carried out. The fractions collected by gel filtration of soluble homogenates of the three strains (571ab, 17bb and Cooper) showed a single peak of Ala AP activity. Partially purified Ala AP of the three strains showed high activity at pH 7.5. The presence or absence of Ca2+ in the assay medium did not produce any difference in activity of Ala AP in the 571ab and Cooper strains, but there was a significant difference in the 17bb strain. The activity of Ala AP in all three strains was essentially unaltered in the presence of inhibitors of serine (PMSF), cysteine (E-64) proteases and carboxypeptidases (pepstatin). Ala AP hydrolyzed alanine amino methylcoumarin (Ala-AMC) maximally, followed by phenyl alanine amino methylcoumarin (Phe-AMC), leucyl amino methylcoumarin (Leu-AMC) and ornithine amino methylcoumarin (Orn-AMC). Ala AP from the three strains showed differential activity towards various substrates. The comparison of alanyl aminopeptidase's activity from different sources is discussed. [source] The identification of a phospholipase B precursor in human neutrophilsFEBS JOURNAL, Issue 1 2009Shengyuan Xu A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be , 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn -1 and sn -2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. [source] Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)FEBS JOURNAL, Issue 24 2007Andre Müller cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5,- and 3,-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH,5His was purified to homogeneity using metal,chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in Km and decreases in kcat values for pyruvate and l -arginine, but had little effect on the Km and kcat values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid,base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329. [source] Two W-containing formate dehydrogenases (CO2 -reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidansFEBS JOURNAL, Issue 11 2003Frank A. M. De Bok Two formate dehydrogenases (CO2 -reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2 -reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U·mg,1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U·mg,1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2 -reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U·mg,1 and 89 U·mg,1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters. [source] The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pHFEBS JOURNAL, Issue 10 2003Gregorio Fernández-Ballester The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ,G = 4.0 ± 0.2 kcal·mol,1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7,9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5,4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family. [source] Myristyl and palmityl acylation of pI 5.1 carboxylesterase from porcine intestine and liverFEBS JOURNAL, Issue 4 2002Tissue, subcellular distribution Immunoblotting analyses revealed the presence of carboxylesterase in the porcine small intestine, liver, submaxillary and parotid glands, kidney cortex, lungs and cerebral cortex. In the intestinal mucosa, the pI 5.1 enzyme was detected in several subcellular fractions including the microvillar fraction. Both fatty monoacylated and diacylated monomeric (F1), trimeric (F3) and tetrameric (F4) forms of the intestinal protein were purified here for the first time by performing hydrophobic chromatography and gel filtration. The molecular mass of these three enzymatic forms was,estimated to be 60, 180 and 240 kDa, respectively, based on size-exclusion chromatography and SDS/PAGE analysis. The existence of a covalent attachment linking palmitate and myristate to porcine intestinal carboxylesterase (PICE), which was suggested by the results of gas-liquid chromatography (GLC) experiments in which the fatty acids resulting from alkali treatment of the protein forms were isolated, was confirmed here by the fact that [3H]palmitic and [3H]myristic acids were incorporated into porcine enterocytes and hepatocytes in cell primary cultures. Besides these two main fatty acids, the presence of oleic, stearic, and arachidonic acids was also detected by GLC and further confirmed by performing radioactivity counts on the 3H-labelled PICE forms after an immunoprecipitation procedure using specific polyclonal antibodies, followed by a SDS/PAGE separation step. Unlike the F1 and F4 forms, which were both myristoylated and palmitoylated, the F3 form was only palmitoylated. The monomeric, trimeric and tetrameric forms of PICE were all able to hydrolyse short chain fatty acids containing glycerides, as well as phorbol esters. The broad specificity of fatty acylated carboxylesterase is discussed in terms of its possible involvement in the metabolism of ester-containing xenobiotics and signal transduction. [source] BJ46a, a snake venom metalloproteinase inhibitorFEBS JOURNAL, Issue 10 2001Isolation, characterization, cloning, insights into its mechanism of action Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4 -reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition. [source] Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporusFEBS JOURNAL, Issue 16 2000Neelam A. Desai An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 °C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA , 3,AMP , RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5,dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5,dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5,dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3, or the 5, end, indicates that C-linkages are resistant to cleavage by nuclease Bh1. [source] Human salivary aggregation in Streptococcus intermedius type g strains: relationship with IgAFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2004Taihei Yamaguchi Abstract Bacterial aggregation is an important step in elimination from the human body to protect against infection. Streptococcus intermedius K1K aggregates in human saliva. In this study, the salivary agglutinin was identified. The aggregation level was very strong in sonic-treated saliva and 1-,m filtrate. Preincubation of human saliva with anti-human , chain serum or anti-human whole saliva serum completely inhibited aggregation, but preincubation with anti-human , chain serum or anti-Fc fragment of human IgG serum had no effect. Agglutinin of human saliva that could aggregate the strain K1K was purified using DEAE,Sepharose CL-6B, Phenyl,Sepharose CL-4B and Sephacryl S200HR gel filtration. Purified salivary agglutinin was characterized with electrophoresis and immunological techniques, indicating that purified material was IgA. Bacterial aggregation was dependent on the presence of calcium. Saliva filtrate specimens from eight healthy men and eight women showed different aggregation activities. Three men and one woman had little activity. These data show that the present bacterial aggregation was an immunoreaction between IgA in saliva and the bacteria dependent on the levels of calcium. In addition, the IgA in human saliva related with possible calcium-dependent antigen(s) on the surface of strain K1K. [source] Lipopolysaccharide binding of the mite allergen Der f 2GENES TO CELLS, Issue 9 2009Saori Ichikawa Lipid-binding properties and/or involvement with host defense are often found in allergen proteins, implying that these intrinsic biological functions likely contribute to the allergenicity of allergens. The group 2 major mite allergens, Der f 2 and Der p 2, show structural homology with MD-2, the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Elucidation of the ligand-binding properties of group 2 mite allergens and identification of interaction sites by structural studies are important to explore the relationship between allergenicity and biological function. Here, we report a ligand-fishing approach in which His-tagged Der f 2 was incubated with sonicated stable isotope-labelled Escherichia coli as a potential ligand source, followed by isolation of Der f 2-bound material by a HisTrap column and NMR analysis. We found that Der f 2 binds to LPS with a nanomolar affinity and, using fluorescence and gel filtration assays that LPS binds to Der f 2 in a molar ratio of 1 : 1. We mapped the LPS-binding interface of Der f 2 by NMR perturbation studies, which suggested that LPS binds Der f 2 between the two large ,-sheets, similar to its binding to MD-2, the LPS-binding component of the innate immunity receptor TLR4. [source] Expression and characterization of ,-glucosidase III in the dwarf honeybee, Apis florea (Hymenoptera: Apoidea: Apidae)INSECT SCIENCE, Issue 4 2007CHANPEN CHANCHAO Abstract Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apis florea. To analyze the developmental stage-specific expression of the ,-glucosidase gene in A. florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction (RT-PCR), it was shown that the highest expression levels of the ,-glucosidase III gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete ,-glucosidase III cDNA was obtained by RT-PCR and sequenced. The 1 701 bp cDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera,-glucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract (0.30 U/mg) sequentially precipitated with 95% saturated ammonium sulfate (0.18 U/mg), and purified by DEAE cellulose ion exchange chromatography (0.17 U/mg), and gel filtration on Superdex 200 (0.52 U/mg). After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band (73 kDa) was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed six matching masses to the A. mellifera (Q17958) and predicted A. florea,-glucosidase III protein with 12% coverage, supporting the probable purification of the same ,-glucosidase III protein as that encoded by the cloned cDNA. [source] Production, purification and thermal characterisation of invertase from a newly isolated Fusarium sp. under solid-state fermentationINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2008Iram Shaheen Summary Production of invertase employing a newly isolated Fusarium sp. under solid-state fermentation was optimised. Different process parameters were optimised. The maximum enzyme activity under optimum conditions was 47.23 ± 2.12 U gds,1 with nitrogen additives. The enzyme was purified by ammonium sulphate precipitation, diethylaminoethyl cellulose ion-exchange chromatography and Sephadex gel filtration. This protocol gave 20.25-fold purification and 5.53% recovery. The optimum pH and temperature for activity were 5.0 and 50 °C. The Km and Vmax values for the enzyme were 8.33 mm and 21.48 ,mol min,1, respectively. A detailed kinetic study of thermal inactivation has been carried out. Enthalpy of activation (,H*) decreased when entropy (,S*) of activation increased at higher temperatures. Moreover, free energy of denaturation (,G*) increased at higher temperature making the enzyme thermally stable. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed. [source] Isolation and Characterization of a Porin-Like Outer Membrane Protein from Xanthomonas campestris pv. campestrisIUBMB LIFE, Issue 1 2002Lingyun Wang Abstract Xanthomonas campestris pv. campestris, a plant-associated pathogenic bacterium, is the causal agent of foliar spots and blights in crucifers. The major outer membrane protein, Omp37, of 37 kDa, has been identified, purified to homogeneity, and its characterization has also been carried out. Native Omp37 behaved as a trimer, as revealed by gel filtration and SDS-PAGE. FTIR measurements revealed a high ,-structure content. The pore-forming ability of the purified Omp37 was studied by the liposome swelling assay. Omp37, to our knowledge, is the first porin that has been isolated from Xanthomonas . This study clearly demonstrates that Omp37 is related to the family of trimeric bacterial porins. [source] Enzyme-Catalyzed Synthesis of a Hybrid N-Linked Oligosaccharide using N-Acetylglucosaminyltransferase IADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2008Rui Chen Abstract The soluble catalytic domain of human N-acetylglucosaminyltransferase I was purified from Escherichia coli and utilized in the enzyme-catalyzed conversion of high mannose N-linked oligosaccharide 1 into the rare hybrid oligosaccharide 2. Analysis of the reaction showed that the conversion of high mannose 1 into hybrid oligosaccharide 2 proceeded to 100% completion as assessed by MALDI-TOF-MS. Purification of the large polar oligosaccharide by gel filtration and silica gel chromatography afforded a 42% isolated yield of oligosaccharide 2. This enzyme-catalyzed reaction can be utilized to produce rare hybrid oligosaccharides for biochemical and structural studies. [source] Isolation and preliminary characteristics of ,- N -acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)JOURNAL OF APPLIED ICHTHYOLOGY, Issue 4 2008B. Sarosiek Summary The aim of this study was to characterize the enzyme ,- N -acetyglucosaminidase (,-NAGase) in the milt and spermatozoa extracts from Siberian sturgeon and rainbow trout. After ion exchange chromatography one protein peak showed ,-NAGase activity in sturgeon milt plasma and sperm extracts of both species. Surprisingly, two protein peaks showing ,-NAGase activity were found in rainbow trout milt plasma. The molecular mass of ,-NAGase was estimated by gel filtration as 127 kDa for rainbow trout spermatozoa, 271 kDa for sturgeon spermatozoa, and 74 kDa for milt plasma from both species. The kinetic parameters were determined for milt plasma and sperm extracts. The optimum pH of the ,-NAGases was 3.8 for sturgeon milt plasma, 4.4 for sturgeon sperm extract, and 4.4,4.8 for milt plasma and sperm extract from rainbow trout. Km value of the ,-NAGases was 0.212, 0.563, 0.779 mm for sturgeon milt plasma, sturgeon sperm extract or rainbow trout extract, respectively. The ,-NAGase from sperm extracts in both species showed 100% activity even after incubation at 56°C by 20 min, whereas its activity was decreased to 23% in sturgeon milt plasma and to 2% in trout milt plasma. [source] Characterization of specific egg yolk immunoglobulin (IgY) against mastitis - causing Staphylococcus aureusJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2008Y.-H. Zhen Abstract Aims:, To evaluate the in vitro activity of egg yolk immunoglobulin (IgY) against mastitis-causing Staphylococcus aureus. Methods and Results:, Specific IgY was produced by immunizing hens with formaldehyde-killed Staph. aureus, using a bacterial strain known to cause mastitis. The IgY, of 94% purity, was obtained from yolks by water dilution, salt precipitations, ultrafiltration and gel filtration. ELISA indicated that the IgY produced was specific to the antigen and five Staph. aureus isolates obtained from mastitic cows. The growth of Staph. aureus was inhibited by specific IgY at concentrations from 1 to 10 mg ml,1 in a dose-dependent manner. The phagocytosis of Staph. aureus by milk macrophages was enhanced in the presence of specific IgY with the highest phagocytic percentage being 30% higher than that without IgY (P < 0·05). Conclusions:, The specific IgY against mastitis-causing Staph. aureus inhibited the growth of Staph. aureus and enhanced the phagocytosis of Staph. aureus by milk macrophages. Significance and Impact of the Study:, Specific IgY would be a potential treatment for bovine mastitis. [source] Penicillium chrysogenum glucose oxidase , a study on its antifungal effectsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2004É. Leiter Abstract Aims:, Purification and characterization of the high molecular mass Candida albicans -killing protein secreted by Penicillium chrysogenum. Methods and Results:, The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (Mr) = 155 000, subunit structure ,2 with Mr,, = 76 000, isoelectric point (pI) = 5·4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1·0 mg ml,1) prevented oxidative cell injuries triggered by 0·004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0·004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX,catalase system was likely to replace the primary inhibition exerted by H2O2. Conclusions:,Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. Significance and Impact of the Study:,Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent. [source] Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffaloJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2001P. Pattnaik Aims:,To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. Methods and Results:,The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4·0,9·0. Conclusions:,The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. Significance and Impact of the Study:,Lichenin could be a potential condidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant. [source] Separation and characterization of the 1,3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild-type and mutant DJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2001H. Malaoui H. MALAOUI AND R. MARCZAK. 2001. Aims:,Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. Methods and Results:,These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. Conclusion:,The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. Significance and Impacts of the study: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol. [source] Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state mediumJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2003Cristina Giatti Marques de Souza The wood-degrading fungus Pleurotuspulmonarius produces at least two laccase isoforms, Lcc1 and Lcc2, when grown on wheat bran solid state medium. The main laccase, Lcc2, was purified to apparent electrophoretic homogeneity by using acetone precipitation, anion-exchange chromatography and gel filtration. Lcc2 had been purified 5.9-fold with a yield of 49%. A specific activity of 19,750 U/mg protein was found using syringaldazine as a substrate under standard assay conditions. The enzyme is a homodimeric glycoprotein containing 44% glycosilation and an apparent molecular mass of 46 kDa. Type I and type III Cu2+ centers were identified by spectrophotometry. The laccase showed optimal activity at pH 6.2,6.5, 4.0,5.5, and 6.0,8.0 with syringaldazine, ABTS and guaiacol as substrates, respectively. For all substrates, the highest oxidation rates were obtained at 50 °C. The enzyme was stable over a large range of pH (4.5,8.0) and at temperatures up to 50 °C. Under standard assay conditions, the apparent KM values were 12, 210 and 550 ,M for syringaldazine, ABTS and guaiacol, respectively. Purified Lcc2 was strongly inhibited by sodium azide, 2-mercaptoethanol and Hg2+, and slightly inhibited by Mn+2 and the chelant agents, EDTA and EGTA. The enzyme was activated by Cu2+ and it retained a high percentage of its activity in the presence of organic solvents, such as acetonitrile and acetone. [source] Analysis of green pit viper (Trimeresurus alborabris) venom protein by LC/MS-MSJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2008Suphan Soogarun Green pit viper venom has major effect on the hematological system having a thrombin-like effect. Thus, this study is designed to analyze the composition of Trimeresurus albolabris venom by performing gel filtration and LC/MS-MS. The purified protein was then digested by trypsin, and the tryptic fragments were analyzed by iontrap spectrophotometry. This study found four types of proteins, namely jerdonitin, stejaggregin-A , chain-1, stejnobin, and stejnihagin-A, as the components of T. albolabris venom. All of these toxins played a greater or lesser role in clot formation or otherwise contributed to cross-reactions in antivenom production. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:225,229, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20232 [source] Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2003DC Simes Abstract Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius,, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5,rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation. [source] Purification and characterization of solvent-tolerant, thermostable, alkaline metalloprotease from alkalophilic Pseudomonas aeruginosa MTCC 7926JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 9 2009Ulhas Patil Abstract BACKGROUND: Microbial proteases are becoming imperative for commercial applications. The protease secreted by Pseudomonas aeruginosa MTCC 7926, isolated from solvent-contaminated habitat was purified and characterized for activity at various edaphic conditions. The purified alkaline protease was investigated for dehairing of animal skin, anti-staphylococcal activity and processing of X-ray film. RESULTS: The protease was 24-fold purified by ammonium sulfate fractionation, sephadex G-100 gel filtration and DEAE-cellulose, with 36% recovery. KM and Vmax, using casein were 2.94 mg mL,1 and 1.27 µmole min,1, respectively. The apparent molecular mass by SDS-PAGE was 35 kDa. Alkaline protease was active at pH 6,11 and temperature 25,65 °C. Its activity was (a) 86.8% in 100 mmol L,1 NaCl, (b) >95% in metal ions (Mn2+, Ca2+, Mg2+, Fe2+) for 1 h, (c) >90% in bleaching agents and chemical surfactants, (d) 135.4 ± 2.0% and 119.9 ± 6.2% with rhamnolipid and cyclodextrin, respectively, (e) stable in solvents for 5,30 days at 27 °C, and (f) inhibited by EDTA, indicating metalloprotein. CONCLUSION: This work showed that purified protease retained its activity in surfactants, solvents, metals, and bleaching agents. The enzyme is an alternative for detergent formulations, dehairing of animal skin, X-ray film processing, treatment of staphylococcal infections and possibly non-aqueous enzymatic peptide synthesis. Copyright © 2009 Society of Chemical Industry [source] | ||||||||||||||||||||||||||||