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 simple method for proteolytic digestion of Coomassie blue-stained proteins in a polyacrylamide matrix is presented. It consists of first reducing and alkylating the stained proteins with dithiothreitol and iodoacetamide in the presence of 0.1% sodium dodecyl sulfate and subsequent digestion with the endoproteinase LysC. The reduction and alkylation step was introduced since experiments with lysozyme and ribonuclease A showed that extremely complex peptide patterns were obtained if no precautions were taken to suppress disulfide bond formation during in-gel digestion of proteins. The advantage of this method is that no blotting step is required for generating internal sequences and that extensive proteolysis occurs which closely resembles that resulting from solution digests. The method has been successfully used to generate internal sequence data from low microgram quantities of proteins excised from 2-dimensional Coomassie blue-stained gels.
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PMID:Internal sequences from proteins digested in polyacrylamide gels. 771 Jan 19

Fourier transform infrared spectroscopy has been used to compare the structure of a range of proteins in solution and in the form of single crystals. An infrared microscope was used to record the spectra of single crystals of the proteins. The proteins studied in this way were hen egg white lysozyme, bovine pancreatic ribonuclease A, bovine gamma-II crystallin, human serum amyloid P component, Endothia parasitica pepsin and Mucor pusillus pepsin. The amide I and amide II bands in the FTIR spectra of these proteins were analysed using derivative procedures thereby providing information on the secondary structure. The crystals were held under a vapour of mother liquor to reduce the effects of dehydration. A comparison of the spectra revealed that spectra recorded from crystals of lysozyme, ribonuclease A and gamma-II crystallin are nearly identical to those recorded from the proteins in solution. However, differences are observed between the spectra of serum amyloid P component, Endothia parasitica pepsin and Mucor pusillus pepsin in solution compared with that of the crystalline form These differences are suggested to be due to rearrangements of turn structures within the protein structure.
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PMID:A comparison of infrared spectra of proteins in solution and crystalline forms. 774 92

A scheme is presented for computing the electrophoretic mobility of proteins in free solution, accounting for the details of the protein shape and charge distribution. The method of Teubner is implemented using a boundary integral formulation within which the velocity distribution, the equilibrium electrical potential around the molecule, and the potential distribution due to the applied field are solved for numerically using the boundary element method. Good agreement of the numerical result is obtained for spheres with the corresponding semi-analytical specialization of Henry's analysis. For protein systems, the method is applied to lysozyme and ribonuclease A. In both cases, the predicted mobility tensors are fairly isotropic, with the resulting scalar mobilities being significantly smaller than for spheres of equal volume and net charge. Comparisons with previously published experimental results for ribonuclease show agreement to be excellent in the presence of a net charge, but poorer at the point of zero charge. The approach may be useful for evaluating approximate methods for estimating protein electrophoretic mobilities and for using electrophoretic measurements to obtain insight into charge distributions on proteins.
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PMID:Computation of the electrophoretic mobility of proteins. 775 31

Although membrane filtration is used extensively to process protein solutions containing a variety of electrolytes, there is currently little fundamental understanding of the effect of the solution environment (and in particular, the solution pH) on the filtrate flux in these systems. We have obtained data for the flux and sieving coefficients during the batch (stirred cell) filtration of solutions of bovine serum albumin, immunoglobulins, hemoglobin, ribonuclease A, and lysozyme through 0.16-micron microfiltration membranes at different pH values. The flux declined significantly for all five proteins due to the formation of a protein deposit on the upper surface of the membrane. The quasi-steady ultrafiltrate fluxes at the individual protein isoelectric pH's were essentially identical, despite the large differences in molecular weight and physicochemical characteristics of these proteins. The flux increased at pH's away from the isoelectric point, with the data well-correlated with the protein surface charge density. These results were explained in terms of a simple physical model in which the protein deposit continues to grow, and thus the flux continues to decline, until the drag force on the proteins associated with the filtrate flow is no longer able to overcome the intermolecular repulsive interactions between the proteins in the bulk solution and those in the protein deposit on the surface of the membrane.
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PMID:Intermolecular electrostatic interactions and their effect on flux and protein deposition during protein filtration. 776 78

Preparative electrooxidation of lysozyme at copper electrodes held at potentials around 1.2 V vs. a saturated calomel reference electrode induces the formation of a yellow chromophore with a concomitant decrease in the pI of the protein. Ion-exchange high-performance liquid chromatography revealed two new lysozyme species with pI values of 10.8 and 10.7 (lysozyme-11.0) which bear the chromophore. Sequence analysis of these two species showed that protein with lower pI was modified at both Tyr 23 and Tyr 20 and the other exclusively at Tyr 23. ribonuclease A, subtilisin BPN', and BSA were also found to produce the same chromophore using similar electrochemical reaction schemes. Characterization of the chromophore by a variety of techniques revealed that it is apparently 3-nitrotyrosine.
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PMID:Electrochemical modification of lysozyme: anodic reaction of tyrosine residues. 776 98

Amino-acetylation and -succinylation reactions in combination with mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations identifying the precise number of acyl groups in partially modified proteins were obtained by electrospray and 252Cf-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecular ions and deuterium exchange experiments provided a relative conformational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivatives. Tryptic peptide mapping analysis, using plasma desorption and fast atom bombardment mass spectrometry, ascertained by mass spectrometric characterization of HPLC-separated modified peptides, yielded the exact identification of acylation sites. Relative reactivities of the amino acylation were derived from the peptide mapping data and from quantitative estimations of modified peptides upon acetylation/trideuteroacetylation and provided direct correlations with the relative surface accessibilities of lysine-epsilon-amino groups taken from X-ray crystallographic structure data of the proteins. The reactive lysine-41 residue in ribonuclease A which is part of the substrate binding site was directly identified from the mass spectrometric data. These results indicate tertiary structure-selective acylation combined with mass spectrometric peptide mapping as an efficient approach for the molecular characterization of surface topology and reactive fundamental lysine residues in proteins.
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PMID:Molecular characterization of surface topology in protein tertiary structures by amino-acylation and mass spectrometric peptide mapping. 787 61

We developed a method for reducing disulfide bonds in proteins under weakly acidic conditions by use of 2-aminothiophenol. The disulfide bonds in hen egg-white lysozyme, ribonuclease A, and soybean trypsin inhibitor were quantitatively reduced by 2-aminothiophenol in phosphate buffer, pH6, containing 8 M Gdn HCl, 1 mM EDTA, and 20% ethanol, for 60 min at 40 degrees C. On analysis of the RP-HPLC patterns of tryptic peptides, which were derived from reduced and S-alkylated lysozyme and ribonuclease A at pH 6, it was confirmed that no side reaction occurred. Moreover, the reduction under weakly acidic conditions was demonstrated to be applicable for the location of such a labile residue as O-acetylated tyrosine.
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PMID:Reduction of disulfide bonds in proteins by 2-aminothiophenol under weakly acidic conditions. 818 36

Denaturations of ribonuclease A, lysozyme, and cytochrome c by guanidine hydrochloride (GdnHCl), urea, and GdnHCl-urea mixture were studied at constant temperature and pH to assess the functional dependence of denaturational free energy change (delta GD) on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl-induced transition curve exhibits a linear plot of delta GD versus [GdnHCl] in the transition zone. To extend delta GD measurements beyond this narrow concentration range, GdnHCl-induced unfolding was measured in the presence of different concentrations of urea. delta GD values from these measurements were corrected for the effect of urea on the free energy change using the appropriate relation. The corrected delta GD data were mapped onto the delta GD versus [GdnHCl] plot. For each protein, the dependence of free energy change on denaturation was found to be linear over the full GdnHCl concentration.
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PMID:A new method for testing the functional dependence of unfolding free energy changes on denaturant concentration. 820 82

51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.
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PMID:Killing of endothelial cells and release of arachidonic acid. Synergistic effects among hydrogen peroxide, membrane-damaging agents, cationic substances, and proteinases and their modulation by inhibitors. 833 Sep 29

6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.
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PMID:"Chitin Leash": a polysaccharide heterobifunctional cross-linking agent which can be cleaved by lysozyme. 837 39


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