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)

Cationic proteins, such as lysozyme, ribonuclease A, and human IgG, impaired the detection of endotoxins with the Limulus amebocyte lysate assay (LAL assay) through formation of endotoxin-protein complexes, demonstrating pronounced masking of endotoxins. Methods, such as phenol extraction, dilution heating, and perchloric acid treatment failed to demask the endotoxins. Also, digestion with trypsin, chymotrypsin, or pronase recovered only 10 to 20% of the applied endotoxins. However, endotoxin recoveries up to 100% were obtained with proteinase K digestion of the samples prior to the LAL assay. This method was then applied to examine the impact of endotoxin masking on endotoxin removal from protein solutions by selective adsorption on membrane adsorbers. It was found that poly-L-lysine and poly(ethyleneimine) as endotoxin-selective ligands were able to pull endotoxins off the proteins studied, thereby guaranteeing successful decontamination.
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PMID:Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins. 960 41

Small-angle x-ray solution scattering (SAXS) is analyzed with a new method to retrieve convergent model structures that fit the scattering profiles. An arbitrary hexagonal packing of several hundred beads containing the problem object is defined. Instead of attempting to compute the Debye formula for all of the possible mass distributions, a genetic algorithm is employed that efficiently searches the configurational space and evolves best-fit bead models. Models from different runs of the algorithm have similar or identical structures. The modeling resolution is increased by reducing the bead radius together with the search space in successive cycles of refinement. The method has been tested with protein SAXS (0.001 < S < 0.06 A(-1)) calculated from x-ray crystal structures, adding noise to the profiles. The models obtained closely approach the volumes and radii of gyration of the known structures, and faithfully reproduce the dimensions and shape of each of them. This includes finding the active site cavity of lysozyme, the bilobed structure of gamma-crystallin, two domains connected by a stalk in betab2-crystallin, and the horseshoe shape of pancreatic ribonuclease inhibitor. The low-resolution solution structure of lysozyme has been directly modeled from its experimental SAXS profile (0.003 < S < 0.03 A(-1)). The model describes lysozyme size and shape to the resolution of the measurement. The method may be applied to other proteins, to the analysis of domain movements, to the comparison of solution and crystal structures, as well as to large macromolecular assemblies.
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PMID:Low-resolution structures of proteins in solution retrieved from X-ray scattering with a genetic algorithm. 963 31

2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native apomyoglobin.
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PMID:Trifluoroethanol stabilizes the pH 4 folding intermediate of sperm whale apomyoglobin. 963 99

Advanced high-resolution NMR spectroscopy, including two-dimensional NMR techniques, combined with high pressure capability, represents a powerful new tool in the study of proteins. This contribution is organized in the following way. First, the specialized instrumentation needed for high-pressure NMR experiments is discussed, with specific emphasis on the design features and performance characteristics of a high-sensitivity, high-resolution, variable-temperature NMR probe operating at 500 MHz and at pressures of up to 500 MPa. An overview of several recent studies using 1D and 2D high-resolution, high-pressure NMR spectroscopy to investigate the pressure-induced reversible unfolding and pressure-assisted cold denaturation of lysozyme, ribonuclease A, and ubiquitin is presented. Specifically, the relationship between the residual secondary structure of pressure-assisted, cold-denatured states and the structure of early folding intermediates is discussed.
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PMID:High-resolution, high-pressure NMR studies of proteins. 964 5

Five stains of Bifidobacterium bifidum (ATCC 11863 and 29591, and NCFB 1453, 1454, 1455) were examined for production of bacteriocins in MRS broth with 0.05% cysteine. Only strain NCFB 1454 excreted a bacteriocin into the broth: it was designated bifidocin B. Bifidocin B was sensitive to several proteolytic enzymes (protease IV, pronase E, protease XVII, proteinase K, trypsin, alpha-chymotrypsin, papain, and pepsin), but was resistant to catalase, peroxidase, lipase, lysozyme, cellulase, ribonuclease A, and amylases. It was also resistant to organic solvents such as ethyl alcohol, acetone, hexane, chloroform, methanol, and ether, and to heating at 90 degrees C for 15, 30, and 60 min or at 121 degrees C for 15 min. Bifidocin B remained active after storage at -20 or -7 degrees C for 3 months and retained biological activity after exposure to pH values of 2 to 10. Bifidocin B was active against some food-borne pathogens and food spoilage bacteria such as Listeria, Enterococcus, Bacillus, Lactobacillus, Leuconostoc, and Pediococcus species but was not active against the other gram-positive and gram-negative bacteria tested. Bifidocin B was produced during exponential phase, reaching a maximum activity of 3,200 AU/ml at early stationary phase. Bifidocin B had a molecular mass of about 3.3 kDa as analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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PMID:Characterization and antimicrobial spectrum of bifidocin B, a bacteriocin produced by Bifidobacterium bifidum NCFB 1454. 970 52

Earlier studies involving water-mediated transformations in lysozyme and ribonuclease A have shown that the overall movements in the protein molecule consequent to the reduction in the amount of surrounding water are similar to those that occur during enzyme action, thus highlighting the relationship among hydration, plasticity, and action of these enzymes. Monoclinic lysozyme retains its crystallinity even when the level of hydration is reduced further below that necessary for activity (about 0.2 gram of water per gram of protein). In order to gain insights into the role of water in the stability and the plasticity of the protein molecule and the geometrical basis for the loss of activity that accompanies dehydration, the crystal structures of monoclinic lysozyme with solvent contents of 17.6%, 16.9%, and 9.4% were determined and refined. A detailed comparison of these forms with the normally hydrated forms show that the C-terminal segment (residues 88-129) of domain I and the main loop (residues 65-73) in domain II exhibit large deviations in atomic positions when the solvent content is reduced, although the three-dimensional structure is essentially preserved. Many crucial water bridges between different regions of the molecule are conserved in spite of differences in detail, even when the level of hydration is reduced well below that required for activity. The loss of activity that accompany dehydration appears to be caused by the removal of functionally important water molecules from the active-site region and the reduction in the size of the substrate binding cleft.
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PMID:Role of water in plasticity, stability, and action of proteins: the crystal structures of lysozyme at very low levels of hydration. 971 62

The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S-S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains.
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PMID:Melting points of lysozyme and ribonuclease A crystals correlated with protein unfolding: a Raman spectroscopic study. 976 18

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.
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PMID:Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins. 978 10

This paper presents a picoliter sample preparation technique utilizing the flow-through principle, allowing on-line coupling of chromatographic systems to be made. The work was performed in order to investigate the characteristics and the physicochemical properties of the sample preparation using typical mobile phase conditions from mu-CLC (column liquid chromatography) separations. The device presented here is a pressure pulse-driven dispenser, formed by two silicon structures processed by conventional micromachining. The pressure pulse is generated in the flow-through channel by a piezoceramic element. Depending on the orifice size, the droplets ejected range between 30 and 200 pL. The maximum ejection frequency is 500 Hz, limited by resonances within the unit. A pyramid-shaped nozzle improves the directivity of the droplets since it reduces the wetting of the orifice front surface area. The risk of particles sticking close to the orifice is also minimized. The analyses of the deposited sample spots were carried out on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer with delayed extraction. It was possible to detect attomole amounts (159-248 amol) of various proteins (cytochrome c, ribonuclease A, lysozyme, and myoglobin) from a single droplet of matrix:analyte 1:1 (drop volume approximately 110 pL). Additionally, it was found that sample enrichment could be carried out using multiple depositions on the same spot; i.e., 31 nM of insulin was easily detected when more than four depositions were made on the same spot, while no detection was possible without sample enrichment. Size optimization of the MALDI sample spot gave target zones of 100-500-micron diameter that matched the size of the laser focal point and resulted in a considerably increased sample throughput.
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PMID:Picoliter sample preparation in MALDI-TOF MS using a micromachined silicon flow-through dispenser. 984 71

Crystallization trials using three polyoxyethylene surfactants as precipitating agents are described. Of the eight soluble proteins screened, five were successfully crystallized at the first attempt. These included lysozyme, catalase, ferritin, ribonuclease A and ubiquitin. Further work suggested that these surfactants could also be suitable for cryo-crystallographic analysis of crystals. At the concentrations used in the crystallization trials [10-40%(v/v)], they are capable of promoting the formation of non-crystalline glasses at cryogenic temperatures (77K). This would facilitate crystal mounting and allow the minimization of crystal irradiation damage. Results from this study also suggest that proteins remain stable at high concentrations of these surfactants [40%(w/v)] and over long time periods (>1 month). A number of membrane proteins were also screened for crystallization. These included photosystems I and II and light harvesting complexes I and II from spinach and bacteriorhodopsin from Halobacterium halobium++. The trial s were unsuccessful both in the absence and presence of heptane-1,2,3-triol and over a wide range of surfactant concentrations.
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PMID:A novel approach for the crystallization of soluble proteins using non-ionic surfactants. 986 32


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