Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method for encapsulating a model protein, lysozyme into hydrophilic uncapped poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres was developed using an oil/water (O/W) single emulsion technique. Lysozyme powder, which was prepared from lyophilization after adjusting a lysozyme solution pH at 3, was molecularly dissolved in a co-solvent system composed of dimethylsulfoxide (DMSO) and methylene chloride. The resulting organic solution containing PLGA was directly emulsified into an aqueous phase, and the organic solvent phase was extracted and evaporated. Various lysozyme-loaded PLGA microspheres having different morphologies were obtained depending on the relative mixing ratio of the two co-solvents used. In vitro release experiments indicated that an initial lysozyme release rate from the microspheres was mainly controlled by ionic interaction between basic amino acid residues in lysozyme and free carboxylate groups in PLGA polymer chain ends, which was probed by incubating the microspheres in a series of media having different NaCl concentrations. However, the protein release leveled off after about 15 days' incubation. To determine the reason for the protein 'no-release' from biodegradable microspheres, a systematic analysis was carried out. By separately adding 0.5 M NaCl, 5 M guanidine HCl, or 5 mM sodium dodecyl sulfate into the release media during the non-release period, it was possible to selectively identify a specific protein non-release mechanism: ionic interaction, non-covalent aggregation, and/or surface adsorption, respectively. It was found that non-covalent aggregation and surface adsorption of lysozyme within the microspheres were the main cause of no further release, whereas ionic interaction between degrading polymer and protein played an insignificant role in the later stage of the release period. The greater amount of additional lysozyme release by sodium dodecyl sulfate than by guanidine hydrochloride suggested that protein surface adsorption was a more critical factor in protein release than aggregation.
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PMID:A new preparation method for protein loaded poly(D, L-lactic-co-glycolic acid) microspheres and protein release mechanism study. 979 50

The oxidation of free tryptophan (Trp) and Trp residues in peptides and proteins by hydrogen peroxide as oxidative agent has been used to evaluate Trp losses and the pattern of degradation products formed. Besides free Trp, four Trp-containing peptides and lysozyme were used as substrates in the aqueous model system. The oxidation rate of Trp and the formation of 16 possible degradation compounds were examined using RP-HPLC and UV, fluorescence, and photodiode array detection. The rate of Trp degradation increased from lysozyme to short-chain peptides to unbound Trp. Only approximately 20% of the total Trp loss could be elucidated by the determined Trp degradation compounds. Oxindolylalanine (Oia), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC), N-formylkynurenine (NFK), dioxindolylalanine (DiOia), kynurenine (Kyn), and 5-hydroxytryptophan (5-OH-Trp) were identified in this order of quantity as degradation compounds, showing the Trp pyrrole moiety to be most susceptible to oxidation. As short peptides such as H-Ala-Trp-Ala-OH were completely hydrolyzed with immobilized Pronase E, Oia and NFK could be identified as main degradation compounds, as could minor amounts of Kyn, DiOia, PIC, and 5-OH-Trp. Acid (6 M HCl), alkaline (4.2 M NaOH), and enzymatic hydrolyses were compared for the determination of Trp degradation compounds in lysozyme. Kyn, Oia, and DiOia could be detected in the hydrogen peroxide treated protein.
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PMID:Oxidation of Free Tryptophan and Tryptophan Residues in Peptides and Proteins. 1055 68

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.
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PMID:Comparison of different methods for the isolation and purification of total community DNA from soil. 1057 2

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.
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PMID:Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. 1078 73

A system of intracellular autolytic enzymes of the bacterium Xanthomonas campestris IBPM B-124 was found to include enzymes with muramidase and glucosaminidase activities, while a system of extracellular bacteriolytic enzymes of the same bacterium includes muramidase, muramoylalanine amidase, and endopeptidase. Using a purification technique including fractional precipitation with ammonium sulfate, gel-filtration on Toyopearl HW-55F, and FPLC ion-exchange chromatography on Mono Q, a preparation of intracellular glucosaminidase was purified 435-fold with 16% yield (SDS-PAGE data indicated the presence of minor protein contaminants). Some physicochemical properties of the purified enzyme were determined: molecular mass 26 kD, Km = 5.6 x 10(-4) M with p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as the substrate, and pH optimum 8.0-8.5. The enzyme is active over a wide range of Tris-HCl buffer concentrations (0.01-0.5 M) and has temperature optimum at 37-40 degrees C. The glucosaminidase activity is sensitive to p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and the disodium salt of ethylenediamine tetraacetic acid (EDTA). The properties of this glucosaminidase markedly differ from those of all extracellular bacteriolytic enzymes of Xanthomonas campestris. These findings indicate that the system of autolytic enzymes of this bacterium functions independently and is not connected with the system of extracellular bacteriolytic enzymes.
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PMID:Intracellular glucosaminidase of the bacterium Xanthomonas campestris IBPM B-124: purification and properties. 1104 95

The use of zymograms in which the bacterial cell wall heteropolymer peptidoglycan is incorporated into the resolving gel of SDS-PAGE has led to the identification of various SDS stable peptidoglycan hydrolases (autolysins). To examine the specificity of autolysins with respect to O-acetylated peptidoglycan, a discontinuous SDS-PAGE system has been developed that operates under neutral conditions. [Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (Bis-Tris) buffers are employed with pH 6.8 and 6.3 for the separating and stacking gels, respectively, while the anode buffer N-2-acetamido-2-hydroxyethanesulfonic acid (Aces)-HCl and the Bis-Tris cathode buffer both had a pH of 6.8. These conditions resulted in a relative trailing ion mobility of 0.349 and 0.137 in the resolving and staking gel, respectively, under room temperature conditions. Peptides and proteins were resolved in the 3-100 kDa range with a 10% acrylamide resolving gel. Comparison of zymograms that incorporated unacetylated or chemically O-acetylated peptidoglycan revealed the specificity of hen egg-white lysozyme for the unacetylated material. A preliminary analysis of the autolysins produced by the urinary tract pathogen Proteus mirabilis indicated that some enzymes were specific for either O-acetylated or non-O-acetylated peptidoglycan while others displayed no clear preference toward either of the two substrates.
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PMID:Differentiation of bacterial autolysins by zymogram analysis. 1126 68

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.
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PMID:Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents. 1174 49

Large scale abstraction and isolation of bacterially synthesized, recombinant-DNA-derived, porcine growth hormone (r-pST) is described. The r-pGH is found in genetic engineering E. coli as the form of inclusion bodies. Pellet fraction which were mainly inclusion bodies, after cell breakage and centrifugation, were collected. Cell envelope components, such as protein, lipid, endotoxin and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. Inclusion bodies were dissolved using 6 mol/L guanidine/HCl and air oxidation is then carried out in the presence of the guanidine/HCl. The Guanidine/HCl protein mixture were diluted by renaturation solution. Guanidine/HCl were removed by dialysis and then correctly refolded, oxidized r-pGH were obtained. Injection experiment of hypophysectomized rats proved r-pST with high native bioactivity was obtained.
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PMID:[Isolation, purification and renaturation of recombinant-DNA-derived porcine somatotropin]. 1191 Jul 69

Investigations are described to extract Se-species from a bacterial sample. The five extraction methods investigated were: hot water, protease, lysozyme, lysozyme-protease, and HCl hydrolysis. The extraction efficiency was determined by comparing the total amounts of selenium in the sample after pressure digestion with the amounts extracted by the different methods described. Efficiencies were found to be only 1% (hot water), ca. 8% (protease, HCl hydrolysis) or ca. 12% (lysozyme, lysozyme-protease). The Se-peak patterns were compared after investigating the extracts with strong anion exchange chromatography-inductively coupled plasma mass spectrometry (SAX-ICP-MS). Most promising were the lysozyme-assisted procedures, which showed the highest diversity of species. Here, in the protease-lysozyme approach, the protease seemed to break down species that had been extracted by lysozyme from the bacterial wall (murein sacculus). The other approaches seemed not to extract many species. Hot water extraction was completely unsuitable, extracting only low amounts of a single, unknown species.
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PMID:Effect of different extraction procedures on the yield and pattern of Se-species in bacterial samples. 1193 31

The pathogenesis of gastric antral hemorrhage and ulceration is unclear. This paper first proposes that antral hemorrhagic ulcers produced in rats are associated with attenuation of defensive parameters (such as mucosal glutathione levels and histamine release, as well as aggravation of aggressive factors) including gastric acid back-diffusion and oxyradical generation. The protective effects of lysozyme chloride and antioxidants on this ulcer model were also evaluated. After being deprived of food for 24 hours followed by refeeding for 1 hour, rats were injected with 1.0 mol HCl/L intragastrically under potent analgesia of diethylether-anesthesia to induce antral ulcer. Control rats received a normal saline solution only. Rats were then given free access to water and food for 4 days. Before the experiment began, rats were again deprived of food for 24 hours. Following anesthetization, their stomachs were irrigated for 3 hours with either normal saline or a physiological acid solution containing 100 mmol HCl/L and 54 mmol NaCl/L. Aggravation of various aggressive and defensive parameters in antral mucosa was observed in refed rats that had received 1.0 mol HCl/L. A high relationship of mucosal glutathione level (r = -0.8754, P <.05) or lipid peroxides generation (r = 0.8198) to antral ulceration was obtained in those ulcerated rats. Intragastric lysozyme chloride (50-200 mg/kg) injected three times daily produced a dose-dependent attenuation of various gastric parameters in the acid-irrigated stomachs of antral ulcer rats. Intraperitoneal injections of various antioxidants, including exogenous glutathione, allopurinol, or dimethylsulfoxide also attenuated antral ulcer. In conclusion, the imbalance of aggressive factors, such as acid back-diffusion and oxyradicals-as well as defensive factors including glutathione and histamine-is important in modulating antral hemorrhagic ulcers that can be ameliorated by lysozyme chloride or antioxidants in rats.
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PMID:Role of acid back-diffusion, glutathione, oxyradical, and histamine in antral hemorrhagic ulcer in rats: the protective effect of lysozyme chloride and antioxidants. 1227 Dec 71


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