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)

An antagonistic strain of Streptococcus faecium was isolated from human feces, and it displayed a marked inhibition of bifidobacteria on agar plates. In liquid culture this isolate produced an antibacterial substance that can be partially purified by ammonium sulfate precipitation followed by gel filtration and ion-exchange chromatography. Its activity was assayed by the inhibition of growth of Bifidobacterium longum. The substance was sensitive to digestion by proteolytic enzymes and alpha-amylase, but was resistant to treatment with 6 M urea, dithiothreitol, 2-mercaptoethanol, ethyl ether, chloroform, and lysozyme. It was also stable to heating at 100 degrees C for 60 min. Its molecular weight was estimated to be about 50,000 by gel filtration.
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PMID:Streptococcus faecium-derived antibacterial substance antagonistic to Bifidobacteria. 741 52

A cDNA fragment of about 860 bp corresponding to the c33-c gene in the non-structural region 3 (NS3) of HCV genome was obtained from one plasma derived from a Chinese HCV carrier who came from Tai' an of Shandong Province, China by the application of reverse transcription (RT) and polymerase chain reaction (PCR) techniques. After the sequence of the cDNA fragment was determined and compared with the equivalent region of the HCV-I (HCV-US) and HCV-II (HCV-BK) genomes, the nucleotide/amino acid sequence homologies were found to be 79.2%/91.3%/ and 91.3%/93.9%, respectively. The prokaryotic expression vector pBV220 was employed for the overproduction of c33-c native recombinant protein in E. coli cells. The expression products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting with antisera of chronic hepatitis C patients, and a molecular weight 31 kD of c33-c viral protein was shown to account for 14% of the total cellular soluble proteins. This product was extracted from the bacterial lysate by lysozyme, Triton X-100 and urea treatment, and purified through ion exchange chromatography. The purified c33-c protein combined with a branch peptide MAP-C-19 representing immunodominant epitopes on the nucleocapsid region of HCV genome was used to develop a Chinese HCV EIA 2nd-generation diagnostic kit for the detection of anti-HCV antibodies. Its specificity, sensitivity and reproducibility were all in keeping with the indexes of the national standard for the quality control of the HCV diagnostic kit. The agreement rate between our kit and Abbott company's HCV EIA second-generation diagnostic kit was 99.33%, and the identified rate of positive anti-HCV of our kit was 2% more than that of the Abbott company's kit.
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PMID:cDNA cloning of c33-c antigen gene derived from NS3 region of Chinese HCV genome, expression in Escherichia coli and development of HCV EIA second-generation diagnostic kit. 752 Jul 1

Four peptides encompassing the entire amino acid sequence of hen lysozyme were examined in aqueous solution and in 50% (v/v) 2,2,2-trifluoroethanol (TFE) by far-UV CD. Two peptides, 1-40 and 84-129, correspond to regions which are helical in the native protein, and together represent the alpha-domain. The beta-domain of the native enzyme was also synthesized as two peptides, one (41-60) containing the residues in the triple stranded antiparallel beta-sheet and the other (61-82) corresponding to a region lacking regular secondary structure. In water at pH 2.0 and 25 degrees C, the monomeric peptides 1-40, 41-60 and 61-82 appear to be predominantly unstructured. By contrast, the peptide 84-129 has considerable, presumably helical structure, corresponding to approximately 19%, or nine residues, on average, which can be unfolded by the addition of 8 M urea or 6 M guanidine hydrochloride. In 50% TFE the conformational properties of the four peptides are again distinct. Although little helical structure is induced in the peptides 41-60 and 61-82, and a native-like extent of helical structure is induced in the peptide 1-40, the peptide 84-29 converts almost entirely to helical structure in 50% TFE. The far-UV CD spectrum of a stoichiometric mixture of the four peptides in water resembles closely that of a denatured state of the intact protein formed by reductive methylation of its four disulphide bonds, but differs significantly from that of the native protein. The far-UV CD spectrum of the peptide mixture in TFE is indistinguishable from that of the intact protein in this solvent, both in the presence and in the absence of its four disulphide bonds. The conformational preferences of the peptides are not predicted using standard assessments of helical propensity or hydrophobicity, but correlate instead with the number of local contacts made in the native protein. On the basis of these results, we suggest that the region 84-129 could play an important role in determining the nature of the early folding events in the folding pathway of the intact polypeptide chain.
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PMID:Conformational properties of four peptides spanning the sequence of hen lysozyme. 756 67

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di- and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains (n > approximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.
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PMID:Immunoassay for the quantification of intracellular multi-ubiquitin chains. 758 72

Proteolysis of hen egg-white lysozyme by thermolysin in 50% aqueous trifluoroethanol for 6-24 h at 40-52 degrees C produces a 'nicked' protein species which was purified to homogeneity by reverse-phase HPLC and characterized. Protein chemistry analytical methods were used to establish that thermolysin cleaves the 129-residue chain of lysozyme at peptide bond Lys97-Ile98. Nicked lysozyme, which is therefore constituted by fragments 1-97 and 98-129 cross-linked by disulfide bonds, was approximately 20% and 60% active towards Micrococcus luteus cells in respect to native intact lysozyme when assayed at 25 degrees C or 5 degrees C, respectively. Circular dichroic measurements provided evidence that nicked lysozyme in aqueous buffer at low temperature maintains the secondary structure content of native lysozyme, whereas the microenvironment of the aromatic chromophores, in particular of tryptophan residue(s), was somewhat perturbed. The stability to heat and urea denaturation of nicked lysozyme was dramatically reduced with respect to that of the intact protein. For example, the tm of the nicked species was 28 degrees C in comparison with 73 degrees C for the unmodified enzyme, both at pH 7.0. Inspection of the X-ray structure of hen lysozyme reveals that thermolysin cleaves at the C-terminus of alpha-helix C (residues 88-98) located at the interface of the two structural domains of the protein, thus destabilizing the helix dipole and disrupting important tertiary interactions of the native enzyme. These results were interpreted considering that lysozyme in 50% aqueous trifluoroethanol is an expanded and flexible protein species largely maintaining native-like secondary structure, but lacking tertiary interactions [Buck, M., Radford, S. E. & Dobson, C. M. (1993) Biochemistry 32, 669-678]. Thus, whereas native lysozyme in its well-packed and rigid structure is quite resistant to proteolysis and only upon thermal unfolding is degraded to many small peptides in an all-or-none process, lysozyme in the trifluoroethanol state is sufficiently flexible to act as a substrate for the protease, but maintains significant secondary structure (helix) precluding extensive proteolytic degradation.
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PMID:Limited proteolysis of lysozyme in trifluoroethanol. Isolation and characterization of a partially active enzyme derivative. 760 52

To increase the folding yield of concentrated reduced lysozyme, we developed a renaturation method by means of dialysis from concentrated urea with redox agents. After lysozyme was incubated in the reducing buffer (8 M urea solution) with oxidized glutathione, renaturation of reduced lysozyme was started by dialysis against the dialyzing buffer containing 8 M urea with redox agents. The urea concentration of the dialyzing bottle was gradually diluted with dialyzing buffer without urea at a flow rate of 0.1 ml/min by high pressure pump. Using this systematic dialysis, a concentration as high as 5 mg/ml of reduced lysozyme could be renaturated in 80% yield, while the folding yield was < 5% even at a concentration of 1 mg/ml using a conventional rapid dilution method [Goldberg et al. (1991) Biochemistry, 30, 2790-2797]. Therefore, it was concluded that gentle removal of urea from denatured proteins, dissolved in concentrated urea solution, by means of dialysis should be useful to renature denatured proteins effectively.
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PMID:Effective renaturation of reduced lysozyme by gentle removal of urea. 763 Aug 89

High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.
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PMID:Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies. 776 97

Acid-soluble extract of human bladder mucosal surface was obtained by washing out the bladder of accidentally dead male human with 1% acetic acid in the presence of proteinase inhibitors. Acid urea polyacrylamide gel electrophoresis (AU-PAGE) indicated that the acid-soluble extract had more than 10 main protein bands, but the currently known antibiotic peptides, e. g., Lysozyme and defensins, were not found in this extract. The quantitation assay of lysozyme showed that lysozyme only amounted to 2.2 microgram per milligram protein. When tested for antibacterial activity by using ultrasensitive radial diffusion assay, the acid-soluble extract effectively killed E. coli ML-35P. The antibacterial activity of the acid-soluble extract was further analysed by gel overlay technique and the result showed that one main protein band, which was referred to as human bladder protein (HBP), was potently antibacterial against E. coli ML-35P. The HBP accounted for 4% of the total acid-soluble extract protein. These results suggest that the presence of antibacterial protein in the bladder mucosa may be the molecular base of the bladder antibacterial defence mechanisms.
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PMID:[Study on antibacterial protein from human urinary bladder mucosa]. 780 84

Reduced and acetylated lysozymes are basic proteins. When their amino groups were variously acetylated and then renatured by sulfhydryl-disulfide (SH-SS) interchange reaction at pH 8.0, the final folding yield decreased as the number of positive charges decreased. The final folding yield of native and Ac1 lysozyme, with one positive charge eliminated, was less sensitive to increasing protein concentration than that of Ac2 lysozyme, where two positive charges had been eliminated. The final folding yield of reduced Ac2 lysozyme increased in the presence of 1 M urea, which reduced the aggregation of unfolded lysozyme. Thus, the aggregation of unfolded lysozymes, which leads to a decrease in the final folding yield, was found to be heavily dependent on their net charges. Moreover, the final folding yield of reduced lysozyme was shown to be increased by use of cystamine as an oxidizing reagent in comparison with 2-hydroxyethyl disulfide or dithiodiglycolic acid. This may support the idea that the final folding yield is influenced by electrostatic interaction between unfolded lysozymes in the early stage of renaturation. In contrast, the concentration dependency of the final folding yield of Ac1 lysozyme was different from those of carboxymethylated His15 and Asp106 lysozymes whose positive net charges were similar to that of Ac1 lysozyme. On the basis of the observations, it is suggested that the formation of the aggregates in the renaturation process might also be affected by the structure of the unfolded state of lysozyme in solution.
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PMID:The role of net charge on the renaturation of reduced lysozyme by the sulfhydryl-disulfide interchange reaction. 785 40

A 14-kDa peroxisomal-matrix protein, named PXP-18, of the yeast Candida tropicalis is a structural and functional homologue of the mammalian nonspecific lipid-transfer protein (identical to sterol carrier protein-2). PXP-18 protected acyl-coenzyme A oxidase (ACO), the rate limiting enzyme of the peroxisomal beta-oxidation of fatty acids, from thermal inactivation at 48 degrees C or 70 degrees C. This effect was dose-dependent and not replaceable either by chicken egg white lysozyme, which is similar to PXP-18 (insofar as it is basic, small, and monomeric), or by bovine serum albumin, a carrier of lipids in the blood. ACO was irreversibly denatured by heat treatment at 70 degrees C for 15 min. However, when ACO and PXP-18 were similarly heat-treated, they formed a large complex at a molar ratio of PXP-18 to ACO subunit that was about one, independent of their initial ratio. This near-stoichiometric complex had ACO activity after a 500-fold dilution and was accompanied by ACO that was free of PXP-18 and indistinguishable from native ACO in size and activity. PXP-18 also protected urate oxidase, another peroxisomal enzyme, from inactivation at 66 degrees C for 15 min and facilitated the renaturation of ACO denatured by 2 M urea. These results indicated that PXP-18 is active in modulating the structure of peroxisomal enzymes in vitro. It is possible that PXP-18 functions as a stress protein or as a part of the system that keeps peroxisomal proteins intact.
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PMID:Near-stoichiometric interaction between the non-specific lipid-transfer protein of the yeast Candida tropicalis and peroxisomal acyl-coenzyme A oxidase prevents the thermal denaturation of the enzyme in vitro. 787 86


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