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1.
The effect of various doses of benzylpenicillin, streptomycin, cephaloridine (ceporin) and exogenic lysozyme on stability of lysozyme binding to lysosomal membranes of human blood monocytes was studied with the procedure developed by the authors which included estimation of the secreted and total lysozyme and evaluation of the stability index of the lysosomal membranes (SILM). Investigation of the changes in the blood monocyte membrane stability was conducted with constant and short-term (60 minutes) exposure to the antibiotics followed by their through elimination. It was noted all the antibiotics increased the SILM which was evident of labilization of the monocyte lysosomal membranes with respect to lysozyme. However, the mechanism of labilization with respect to various antibiotics was different judging from the amount of the secreted and total lysozyme. Labilization under the effect of benzylpenicillin and streptomycin was most probably due to changes in the lysozyme functional activity in the presence of these antibiotics and that under the effect of exogenic lysozyme was probably due to penetration into the monocytes and simultaneous impairment of the monocyte secretory activity. Cephaloridine had the labilizing effect by inhibition of the lysozyme intracellular synthesis.  相似文献   

2.
Bindings of calcium to lysozyme and its derivatives were studied by UV difference spectroscopy at various pH's. The binding constant was ca. 40 m-1 at around neutral pH. The binding caused proton release from lysozyme and did not inhibit the binding of tri-N-acetylglucosamine to lysozyme. In the presence of 0.2 M Ca2+, lysozyme showed 26% of the activity of the free enzyme toward hexa-N-acetylglucosamine but the cleavage pattern was similar to that of the free enzyme. Thus, calcium was predicted to bind near the catalytic carboxyls to cause inhibition of lysozyme activity. It was found from the results of protease digestion that calcium binding shifted the native-denatured transition in lysozyme toward the native state.  相似文献   

3.
Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.  相似文献   

4.
The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.  相似文献   

5.
The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.  相似文献   

6.
Animals and plants both respond rapidly to pathogens by inducing the expression of defence-related genes. Within this context, a prominent role has been assigned to the lysozyme. In the present study we isolated and carried out detailed analysis of the lysozyme gene in the plant nematode Meloidogyne artiellia. The expression of lysozyme was up-regulated following exposure of M. artiellia juveniles to the Gram-negative bacterium Serratia marcescens. On the other hand, when isolated eggs containing embryos at various developmental stages were challenged with bacteria, no increase in lysozyme expression was detected. Evidence of lysozyme expression regulation was obtained in the case of adult male and females worms collected from soil. The lysozyme gene was expressed solely in the nematode intestine and, as it is predicted to be secreted, may protect the nematode from microbial infections originating in the intestinal lumen or in the pseudocoelom. This paper demonstrates, to our knowledge for the first time, the immune response to infection in a plant parasitic nematode.  相似文献   

7.
Using commercial anti-lysozyme antibodies and anti-dimerized lysozyme rabbit serum produced by us we demonstrated by immunohistochemistry, in some organs of rats, the expression of exogenous egg white lysozyme preparations beside the native lysozyme. After oral administration, the egg white lysozyme was detected in intestinal epithelium, proximal and distal tubules of some nephrons, pulmonary alveolar walls and hepatocytes in the 3rd zone of liver acini, whereas native lysozyme was strongly expressed in intestinal and pulmonary macrophages in both the experimental and control animals. However, expression of the dimerized lysozyme released from the intraperitoneally implanted mini-osmotic pumps and detected using specific antisera was evident only on erythrocytes in intestinal blood vessels. It is concluded that the lysozyme preparations administered per os or parenterally are resorbed to blood circulation and distributed among various organs in an active form and maintaining their antigenic specificity. It may speak for their direct anti-inflammatory and immunomodulatory effects in respiratory, urinary, digestive and other systems.  相似文献   

8.
Lysozyme (mucopeptide-N-acetylmuramylhydrolase, EC 3.2.1.17) is present in mammalian cartilage. Lysozyme was isolated and purified from bovine and canine cartilage and from dog serum using various chromatographic steps and affinity chromatography on carboxymethylated chitin. Amino acid analysis of bovine cartilage lysozyme showed that it is similar to other mammalian lysozymes. Anti-canine lysozyme antibodies cross-react with calf lysozyme, but not with hen egg white or embryonic chick cartilage lysozyme. In the epiphyseal plate of the dog, 90-μm sections were analyzed for lysozyme and its was found that in the hypertrophic zone its concentration is approximately six times higher than it is in the resting zone. Using immunocytochemical techniques at the electromicroscopic level, lysozyme in the epiphyseal plate of the dog was localized extracellularly, mainly in the immediate vicinity of the chondrocytes, the territorial matrix.  相似文献   

9.
The interaction of SDS with lysozyme was analyzed with enzyme activity and with NMR, fluorescence, and UV difference spectroscopies using various alkyl sulfates and variously modified lysozymes. SDS formed a stable complex with lysozyme without causing a gross conformational change in the enzyme molecule. Some SDS molecules bound to the active site cleft of lysozyme and therefore strongly inhibited the activity of lysozyme. Hydrophobic regions and positive charges for protein side, and a hydrophobic tail (possibly more than 8 carbons in alkyl chain) and a negative charge for detergent side were required for the formation of the complex.  相似文献   

10.
Cells of Anacystis nidnlans were grown in synchronous culture using a light-dark alternation to obtain synchronization. Two synchronous cycles were obtained with, decay of synchrony beginning with the third cycle. Cells of various ages in the growth cycle were treated with lysozyme to form spheroplasts. The percentage of spheroplast formation varied with age of the cells. After extended periods of lysozyme treatment, up to 90% of the cells of all ages showed spheroplast formation. Some cells were resistant to the action of lysozyme regardless of age or length of treatment. An ultrastructure study of the spheroplast was made. The electron-dense inner layer of the cell wall was removed by the action of lysozyme on the glucosamine residues of the cell wall, indicating true spheroplast formation. The photosynthetic apparatus became more pronounced with extended treatment with lysozyme.  相似文献   

11.
Fractionation of the highly purified but low active recombinant protein destabilase-lysozyme (Dest-Lys) by cation exchange chromatography on a TSK CM 3-SW chromatography the non-active fraction (IV) containing 90% of total protein has been separated. Fractions I, II, and III contained proteins with lysozyme and isopeptidase activities and their lysozyme activity correlated with the activity of native Dest-Lys. However, the ratio of lysozyme and isopeptidase activities differed in these fractions; maximal lysozyme activity was found in fraction III, while maximal isopeptidase activity was associated with fraction I. Possible regulation of different functions of Dest-Lys is discussed in the context of formation of its various complexes.  相似文献   

12.
Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.  相似文献   

13.
The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3.  相似文献   

14.
Factors affecting the activity of human lysozyme (EC 3.2.1.17) toward cell suspensions of Micrococcus lysodeikticus were reexamined. Effects of substrate concentration, pH, and ionic strength and matrix effects of protein were assessed with special emphasis on the interdependence of various parameters. On the basis of these evaluations, an optimized kinetic turbidimetric method for lysozyme assay was set up. The method was applied for automation with a System Olli 3000 analyzer. The new automated lysozyme assay proved good for routine clinical use in regard to analysis speed, sensitivity, linearity, and reproducibility. Reference values for serum, urinary, and cerebrospinal fluid lysozyme were assessed with the automated method.  相似文献   

15.
Yang  Wenli  Zhang  Nan  Wang  Qiang  Wang  Ping  Yu  Yuanyuan 《Bioprocess and biosystems engineering》2020,43(9):1639-1648

Lysozyme, a type of natural enzyme, has been widely used for bacteriostatic functionalization of various materials due to its efficient and selective antibacterial properties. Herein, we report the preparation and characterization of an eco-friendly antibacterial textile based on the immobilization of lysozyme from chicken egg white onto wool fibers. Tris(hydroxymethyl)phosphine (THP) was employed as the cross-linker for the immobilization of lysozyme on the surface of wool fiber. The mechanism of THP cross-linking was investigated via phosphorus test, energy-dispersive spectroscopy (EDX) and Fourier transform infrared spectroscopy (FT-IR). The surface staining, optimization of immobilization parameters, morphology, antibacterial properties, and durability of wool fibers with immobilized lysozyme were also assessed. The results show that hydroxymethyl groups of THP reacted with amino groups of wool fiber and lysozyme through Mannich reaction, which successfully immobilized lysozyme on the wool fiber. The wool fibers incorporated with lysozyme had better antibacterial properties and durability compared with the untreated wool fabric. This facile immobilization approach of lysozyme provides an effective strategy for environmentally benign modification and functionalization of keratin and keratin-containing materials.

  相似文献   

16.
Abstract Recent studies carried out by our group suggest that lysozyme binds to bacterial lipopolysaccharide with a high affinity to produce a complex, and inhibits various biological activities of lipopolysaccharide. Although the basic structure of lipopolysaccharide is independent of the species and strains of Gram-negative bacteria, many structural factors such as O-antigenic polysaccharide, lipid A, substituted groups, and associated molecules, affect the biological activities of lipopolysaccharide. In this study, we prepared lysozyme/lipopolysaccharide complexes using various structures of lipopolysaccharide and compared the activity and physiochemical properties. Native and dansylated lysozyme were found to bind to all tested lipopolysaccharides. The mitogenic activity and TNF production by all tested lipopolysaccharides were significantly reduced by complex formation in vitro. Administration of the complex prepared by various lipopolysaccharides produced significantly less quantities of TNF in the septic shock model. These results suggested that binding of lysozyme to lipopolysaccharide is important for the host both in pathophysiological responses to lipopolysaccharides and in the modification of lipopolysaccharide biological activity.  相似文献   

17.
Summary The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles. Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria infected with T4 gene 25 amber mutants. The 15K lysozyme was also found to be expressed in E. coli B cells transformed with a plasmid containing only a small portion of the T4 genome but which included T4 gene 25. These observations indicate that the 15K lysozyme is the gene 25 product.  相似文献   

18.
The time dependence of aggregation in supersaturated lysozyme solutions was studied using pulsed-gradient spin-echo NMR diffusion measurements as a function of lysozyme concentration at pH 6.0 and 298 K in the presence of 0.5 M NaCl. The measurements provide estimates of the weight-averaged diffusion coefficient of the monomeric to intermediate molecular weight lysozyme species present in the solution (very large aggregates and crystals are excluded from the average due to the NMR relaxation-weighting effects inherent in the method). The results show that the average molecular weight of the various lysozyme aggregates changed with sigmoidal kinetics and that these kinetics were strongly influenced by the initial lysozyme concentration. The visualization of the time dependence of the protein aggregation afforded by this method provides a deeper understanding of how the crystallizing conditions (especially the initial protein concentration) are related to the resulting crystals.  相似文献   

19.
Detailed characterization of enzyme susceptibility of bacterial cellulose containing N-acetylglucosamine (GlcNAc) residues (N-AcGBC) which possess high susceptibility for cellulase and lysozyme and slight susceptibility for chitinase was studied. Turbidimetric lysozyme assay of N-AcGBC showed that (i) the susceptibilities of various N-AcGBCs for lysozyme were proportional to GlcNAc content, and (ii) N-AcGBC homogenates were divided into two groups based on the rate of turbidity reduction (not dependent on GlcNAc content). High reactivity of N-AcGBC for lysozyme would arise from fine microfibrils characteristic of bacterial cellulose (BC) and random distribution of GlcNAc residues in N-AcGBC because water soluble oligomers of N-AcGBC produced by lysozymic hydrolysis did not inhibit lysozyme activity; however, the random distribution of GlcNAc seemed to result in the slight susceptibility of N-AcGBC for chitinase. The rate of cellulolytic turbidity reduction of N-AcGBC was slower than that of BC, which arose from the inhibition for binding of cellulase by GlcNAc residues.  相似文献   

20.
Ethanol is used to precipitate proteins during various processes, including purification and crystallization. To elucidate the mechanism of protein precipitation by alcohol, we have investigated the solubility and structural changes of protein over a wide range of alcohol concentrations. Conformation of hen egg-white lysozyme was changed from native to α-helical rich structure in the presence of ethanol at concentrations above 60%. The solubility of lysozyme was reduced with increasing ethanol concentration, although gel formation at ethanol concentrations between 60% and 75% prevented accurate solubility measurements. SH-modified lysozyme showed largely unfolded structure in water and α-helical structure in the presence of ethanol. More importantly, solubility of the chemically modified lysozyme molecules decreased with increasing ethanol concentration. There is no indication of increased solubility upon unfolding of the lysozyme molecules by ethanol, indicating that any favorable interaction of ethanol with the hydrophobic side chains, if indeed occuring, is offset by the unfavorable interaction of ethanol with the hydrophilic side chains and peptide bonds.  相似文献   

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