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 automated Minigel electrophoresis system (PhastSystem, Pharmacia, Uppsala, Sweden) was tested for human tear protein analysis. Tear samples were treated under nonreducing or reducing conditions before sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE). Micro-amounts of tears (2 microliters) were sufficient for analysis and separation and visualization of proteins were completed within 2 hr. Tear proteins were identified using purified control proteins and immunoblotting techniques, using antisera against immunoglobulin (Ig) A (alpha) heavy chains, Ig heavy and light chains, secretory components and lactoferrin. In nonreduced tears, lactoferrin (seen as a double band), serum albumin, tear-specific prealbumin (TSPA), and lysozyme were clearly separated. Secretory IgA (sIgA) was seen as a smear on top of the gel. Immunostaining also showed a major Ig light chain containing protein. After reduction, the protein profiles showed marked changes. In reduced tears, immunoglobulin heavy and light chains (molecular weight [MW]: 64 and 28 kD, respectively) were detected on the SDS-PAGE profile after immunostaining, and represented disulfide cleavage fragments, which originated from sIgA. Reduction resulted in the liberation of the secretory component piece (MW: 85 kD), which was found to co-migrate with the tear lactoferrin bands. Both lactoferrin and serum albumin acted as larger proteins on SDS-PAGE after reduction. The authors found that the two methods of sample treatment, before electrophoresis, resulted in marked differences on the electropherograms.
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PMID:SDS-Minigel electrophoresis of human tears. Effect of sample treatment on protein patterns. 199 90

We investigated protein accumulation on disposable extended wear contact lenses. Fifteen volunteers were fit with one low water content, non-ionic lens (Bausch & Lomb's SeeQuence) randomly assigned to one eye and a high water content ionic lens (Vistakon's Acuvue) assigned to the fellow eye. During the first 7 weeks of extended wear the lenses were removed weekly for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein deposition, and replacement lenses were inserted. Four subjects completed additional test sessions of 1 minute, 15 minutes, 24 hours, and 1 week extended wear. Lysozyme accumulation, as measured by SDS-PAGE, increased with wearing times up to one week on all Acuvue lenses, but after 24 hours wear lysozyme accumulation did not increase on the SeeQuence lens. Proteins falling into the reported molecular weight ranges of albumin, PMFA, IgG, IgA (sec), lactoferrin and subunits of protein G were evident on all gels at 1 minute of wear, but these protein groups did not have a detectable increase in deposition after 24 hours wear for either the SeeQuence or the Acuvue lenses. In most cases, the protein accumulation evident from SDS-PAGE analysis was not observable by biomicroscopy using standard clinical methods. A few patients reported preference for the initial comfort and vision achieved by the Acuvue lens, but no preference was found after adaptation.
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PMID:Protein accumulation on disposable extended wear lenses. 200 85

To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions. Some mutant lysozymes were found to form a compact conformation with native-like mobility in the presence of SDS. The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116----Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.
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PMID:Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion. 204 Mar 7

Plasmid pBR322 and its derivative containing strong promoter phi 10 of bacteriophage T7 RNA polymerase were used as vectors. A fragment of bacteriophage T7 DNA which was digested with two restriction endonucleases (AvaII and HaeIII) was cloned in the BamHI site of plasmid pBR322 and its derivative pAR951, respectively. The inserted DNA is a segment of 632 base pairs containing the complete coding sequence of both T7 gene 3.5 and weak promoter phi 3.8 for bacteriophage T7 RNA polymerase. The function of T7 gene 3.5 is known to code for bacteriophage T7 lysozyme. Transformants that carry the recombinant plasmid were tested for intracellular lysozyme by adding CHCl3. Both cloned strains produce active T7 lysozyme. The gene product, T7 3.5 protein, was analyzed by 10 -20% gradient polyacrylamide- SDS electrophoresis. The result showed that the expression of inserted T7 gene 3.5 in pBR322 derivative is stronger than that in pBR322.
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PMID:Cloning of the bacteriophage T7 lysozyme gene. 210 4

PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].
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PMID:Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes. 215 85

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.
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PMID:Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase. 216 79

Five antimicrobial peptides were extracted from the cytoplasmic granules of rabbit neutrophils with 0.01 mol/L citric acid. SDS-PAGE gel densitometric analysis of the citric acid extract showed that they accounted for 34%-42% of the total extract proteins. When subjected to acid urea polyacrylamide gel electrophoresis, five peptides migrated much further to the cathode than lysozyme. Beginning with the most cathodal component, they were in turn referred to as NP1, NP2, NP3, NP4, and NP5. They were purified from the citric acid extract by virtue of preparative polyacrylamide gel electrophoresis. Each of them was of low molecular weight (2,500-6,000) as determined by SDS-PAGE. Three of the peptides were tested in vitro for fungicidal activity. When Candida albicans (10(7)/ml) were incubated with 12.5 micrograms/ml of NP1 or NP2 at 37 degrees C for 2 h, the death rates of the organisms were 96.6% and 93.3% respectively. NP3 was shown to be active against Candida albicans although less potent than NP1 and NP2.
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PMID:[Purification and identification of antimicrobial peptides of rabbit neutrophils]. 219 31

A simple extraction procedure was used for preparing cell surface proteins (CSPs) from Shigella dysenteriae type 1. The preparations obtained using either buffer or water extractions were free from lipopolysaccharide (LPS), as well as cytoplasmic and periplasmic proteins. By SDS-PAGE, about 25 polypeptides were detected, and Western-blot analysis recognised 15 polypeptide antigens. When analysed by crossed immunoelectrophoresis, using anti-Shigella dysenteriae type 1 rabbit sera, 18 antigenic bands were identified. Proteins obtained by this method were found to be highly immunogenic in rabbits. The cell-surface proteins were compared to outer membrane proteins (OMPs) obtained from the S. dysenteriae type 1 strain by a standard procedure involving lysozyme-EDTA extraction, sucrose density centrifugation, and detergent treatment. They were found to contain periplasmic, cytoplasmic, and lipopolysaccharide contaminants. Thus, the procedure described here offers a quick and simple alternative for obtaining relatively pure cell surface proteins from Shigella dysenteriae type 1. This method will be useful when immunogenically active proteins free from other cellular components are required for studies.
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PMID:Cell surface proteins from Shigella dysenteriae type 1. 220 98

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 degrees C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.
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PMID:Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods. 221 83

1. Tests for glycosidases were performed in homogenates of Brachionus plicatilis. 2. Hydrolytic activity was detected with the following substrates: (a) with synthetic substrates (NP = 4-nitrophenyl): NP-alpha- and NP-beta-D-glucopyranoside, NP-alpha- and NP-beta-D-galactopyranoside, NP-N-acetyl-beta-D-glucosaminide, NP-N-acetyl-beta-D-galactosaminide, NP-alpha- and NP-beta-D-mannopyranoside and NP-alpha-L-fucopyranoside; (b) with disaccharides: sucrose, maltose, trehalose, isomaltose, cellobiose, gentiobiose and lactose; (c) with polysaccharides: laminarine, carboxymethyl-cellulose, avicel, Micrococcus luteus (for lysozyme) and 4-nitrophenyl-alpha-D-maltoheptaoside (for amylase). 3. The pH dependence of the glycosidase activities was determined. 4. The distribution of enzyme activities within fractions from the homogenate was studied in order to localize them within the cell. 5. Proteins from Brachionus homogenate were separated by SDS-gel electrophoresis and the positions of the following glycosidase activities were detected by assays performed on the gels (estimated molecular weights in parentheses): alpha-glucosidase (250,000); beta-glucosidase (200,000); beta-galactosidase (70,000); N-acetyl-beta-glucosaminidase (60,000).
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PMID:Glycosidases in Brachionus plicatilis (Rotifera). 232 73


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