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 improved method is described for the purification of the DNA-dependent RNA polymerase [ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6] from Escherichia coli. The method involves lysozyme-sodium deoxycholate lysis, low-speed centrifugation, precipitation with Polymin P, elution from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DNA-cellulose and Bio-Gel A 5m. RNA polymerase is purified to electrophoretic homogeneity in 2 days with a recovery of 45%, resulting in a yield of 250 mg of holoenzyme from 500 g of cells.
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PMID:A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. 110 52

Anion exchange membranes prepared by adsorption of polymers on Formed-In-Place microfiltration substrates were formed and ion-exchange separations of solutions containing two proteins were determined by ion exchange membrane sequential separation procedures, similar to affinity membrane separation procedures. Representative ion exchange separation processes utilizing adsorbed poly(ethylene imine) (PEI) as the ion exchange membrane for the separation of the components of solutions containing two proteins, bovine serum albumin (BSA) and lysozyme and ovalbumin and lysozyme, are described. The stability of the PEI adsorbed layer, binding characteristics of the BSA to the membrane and purification properties of the procedure were determined.
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PMID:Selective protein separations using Formed-In-Place anion exchange membranes. 136 50

The cationic ultrastructural tracer polyethyleneimine (PEI: pI approximately equal to 11.0), binds electrophysically to uniformly spaced discrete electron-dense anionic sites present in the laminae rarae of the rat glomerular basement membrane (GBM), mesangial reflections of the GBM, Bowman's capsule, and tubular basement membranes when administered intravenously. Computer-assisted morphometric analysis of glomerular anionic sites reveals that the maximum concentration of stainable lamina rara externa (lre) sites (21/10,000 A GBM) occurs 60 minutes after PEI injection with a site-site interspacing of 460 A. Lamina rara interna (lri) sites similarly demonstrate a maximum concentration (20/10,000 A GBM) at 60 minutes with a periodicity of 497 A. The concentration and distribution of anionic sites within the lri was irregular in pattern and markedly decreased in number, while the lre possesses an electrical field that is highly regular at all time intervals analyzed (15, 30, 60, 120, 180, 240, and 300 minutes). Immersion and perfusion of renal tissue with PEI reveals additional heavy staining of the epithelial and endothelial cell sialoprotein coatings. PEI appears to bind to glomerular anionic sites reversibly: ie, between 60 and 180 minutes the concentration of stained sites decreases. At 300 minutes, the interspacing once again approaches the 60-minute concentration. This suggests a dynamic turnover or dissociation followed by a reassociation of glomerular negatively charged PEI binding sites. In contrast, morphometric analysis of anionic sites stained with lysozyme and protamine sulfate reveals interspacings of 642 A and 585 A, respectively; in addition, these tracers produce major glomerular ultrastructural alterations and induce transient proteinuria. PEI does not induce proteinuria in rats, nor does it produce glomerular morphologic alterations when ten times the tracer dosage is administered intravenously. These findings indicate that the choice of ultrastructural charge tracer, the method of administering the tracer, and the time selected for analysis of tissue after administration of tracer significantly influences results. Morphometric analysis of the distribution of glomerular anionic sites in nonproteinuric rats provides a method of evaluating quantitative alterations of the glomerular charge barrier in renal disease models.
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PMID:Glomerular anionic site distribution in nonproteinuric rats. A computer-assisted morphometric analysis. 407 20

An improved purification procedure is described for the rho transcription termination factor of Escherichia coli. The method involves lysozyme--sodium deoxycholate lysis, Polymin P fractionation, and chromatography on phosphocellulose, poly(uridylic acid)--Sepharose, and AMP--agarose. The method yields up to 9 mg of electrophoretically pure protein from 200 200 g of E. coli MRE 600. From quantitative amino acid analysis rho is calculated to have an E280nm (1%) of 3.7 +/- 0.3. The purified rho has an ATPase specific activity of 32 nmol of Pi released min-1 microgram-1 when poly(cytidylic acid) is used as a cofactor, and it functions effectively in termination of T7 DNA transcription. A subunit molecular weight of 48 000 for rho was determined by phosphate-buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The amino acid composition and circular dichroism spectrum in the far-ultraviolet for rho are presented.
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PMID:Procedure for purification of Escherichia coli ribonucleic acid synthesis termination protein rho. 645 99

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pI. Four polycationic polymers were examined: polyethylenimine (PEI; MWav = 15000), Polybrene (MWav = 25000), poly(methoxyethoxyethyl)ethylenimine (MWav = 64000), and poly(diallyldimethylammonium chloride) (MWav = 10000). Detection of proteins with high pI was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethyl-ammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic flow was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene. This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings. A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Zp(0) = +7.6 +/- 0.1) and established the acidity of the alpha-ammonium group of lysozyme (pKa = 7.8 +/- 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.
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PMID:Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins. 910 78

The selective interaction between polyelectrolyte multilayers (PEM) consecutively adsorbed from poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAC) and a binary mixture containing concanavalin A (COA) and lysozyme (LYZ) based on electrostatic interaction is reported. The composition and structure of the PEM and the uptake of proteins were analyzed by in situ attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy, and the morphology and thickness were characterized by atomic force microscopy (AFM) and ellipsometry. The PEM dissociation degree and charge state and the protein adsorption were shown to be highly dependent on the outermost layer type and the pH in solution. High protein uptake was obtained under electrostatically attractive conditions. This was used to bind selectively one protein from a binary mixture of LYZ/COA. In detail it could be demonstrated that six-layered PEM-6 at pH = 7.3 showed a preferential sorption of positively charged LYZ, while at PEM-5 and pH = 7.3 negatively charged COA could be selectively bound. No protein sorption from the binary mixture was observed at pH = 4.0 for both PEM, when COA, LYZ, and the outermost PEI layer of PEM-5 were positively charged or the outermost PAC layer of PEM-6 was neutral. Furthermore, from factor analysis of the spectral data the higher selectivity was found for PEM-5 compared to PEM-6. Increasing the ionic strength revealed a drastic decrease in the selectivity of both PEM. Evidence was found that the proteins were predominantly bound at the surface and to a minor extent in the bulk phase of PEM. These results suggest possible working regimes and application fields of PEI/PAC multilayer assemblies related to the preparative separation of binary and multicomponent protein mixtures (biofluids, food) as well as to the design of selective protein-resistant surfaces.
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PMID:pH dependence and protein selectivity of poly(ethyleneimine)/poly(acrylic acid) multilayers studied by in situ ATR-FTIR spectroscopy. 1660 51

In the aim of protecting stainless steel surfaces against protein and/or bacterial adhesion, thin films including the glycosidase hen egg white lysozyme (HEWL) and/or the synthetic polymer poly(ethylene glycol) (PEG) were covalently coated onto flat substrates by wet chemical processes. Chemical grafting of both species was carried out by covalent binding to surfaces pretreated by the polyamine poly(ethylene imine) (PEI). Surfaces were characterized at each step of functionalization by means of reflection-absorption infrared spectroscopy by modulation of polarization (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to determine the atomic and molecular composition of the interfaces, respectively. Then, the ability of the so-modified surfaces to prevent protein adsorption and bacterial adhesion together with their biocide properties were demonstrated by three local tests employing bovine serum albumin (BSA), and the bacteria Listeria ivanovii and Micrococcus luteus. A new test was implemented to assess the local enzymatic properties of HEWL. Cografting of PEG and HEWL resulted in a surface with both antiadhesion and antibacterial properties.
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PMID:Grafting of lysozyme and/or poly(ethylene glycol) to prevent biofilm growth on stainless steel surfaces. 1916 31

Herein, we report two simple label-free electrochemical aptasensors for protein detection using layer-by-layer (LBL) self-assembled multilayers with ferrocene-appended poly(ethyleneimine) (Fc-PEI), carbon nanotubes (CNTs) and aptamer. In one sensing strategy, the Fc-PEI, CNTs and DNA aptamer are LBL assembled on the electrode surface via electrostatic interaction. In the presence of target, the aptamer on the outermost layer of the LBL self-assembled multilayer would catch the target on the electrode interface, which makes a barrier for electrons and inhibits the electro-transfer, resulting in the decreased DPV signals of Fc-PEI. Using this strategy, a wide detection range (0.3-165 ng ml(-1)) for model target thrombin is obtained, with a low detection limit of 0.14 ng ml(-1). In the similar sensing strategy for detection of lysozyme, a wide detection range (0.2 ng ml(-1) to 1.66 microg ml(-1)) and a low detection limit (0.17 ng ml(-1)) are obtained. These results prove that the LBL sensing strategies developed possess sensitivity, selectivity, stability and generality.
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PMID:Layer-by-layer electrochemical biosensor with aptamer-appended active polyelectrolyte multilayer for sensitive protein determination. 2010 54

Protein stability assessment of seven model proteins in the presence of low molecular weight polyethyleneimine (PEI, MW 2000 Da) was performed. Thermodynamic stability, monitored by circular dichroism (CD) spectroscopy, showed that the polymer did not have a major effect on the melting temperature (T(m)) of the basic proteins - muscle lactate dehydrogenase (LDH), ribonuclease A, lysozyme and cutinase, while for the acidic ones - human growth hormone, human serum albumin and heart lactate dehydrogenase - there was a shift in T(m) towards lower temperatures. The secondary structures of the basic proteins were essentially the same, with none or a slight increase in the CD spectra, in presence of the polymer. In the case of the acidic proteins, the CD spectra were diminished mostly due to phase separation. Assuming a homogeneous distribution of the net charge on the protein surface a quantitative inverse relationship was established between surface charge density of the acidic proteins and the PEI(2000) concentration required for maximum flocculation. Despite lowering the thermal stability of acidic proteins, PEI(2000) was seen to protect heart LDH at an increasing oxidative stress.
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PMID:Polyethyleneimine-protein interactions and implications on protein stability. 2041 30

Two hydrolytic enzymes, namely lysozyme and trypsin, were covalently immobilized onto stainless steel surfaces using wet chemistry processes. The immobilization strategy took advantage of the spontaneous physisorption of the polymer poly(ethylene imine) (PEI) onto stainless steel to yield a firmly attached, thin organic layer containing a high density of primary amine functions. Both enzymes were then covalently grafted to the surface via a glutaraldehyde cross-linker. Alternatively, a thicker underlayer of PEI was chemisorbed by cross-linking two PEI layers by glutaraldehyde. The effective presence of both enzymes on the stainless steel surfaces and their relative amount were assessed by immunochemical assays employing specific anti-enzyme antibodies. Eventually, the hydrolytic activity of the immobilized enzymes was evaluated by local enzymatic tests with suitable substrates. This work demonstrates that, although the amount of enzymes did not vary significantly with the underlayer thickness, their hydrolytic activity could be much improved by increasing the distance from the oxide surface and, likely, by favoring their accessibility. Our data suggest that the immobilization of enzymes on solid oxide surfaces is feasible and efficient, and that the enzymes retain catalytic activity. It may thus provide a promising route towards biofilm-resistant materials.
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PMID:Bioengineering of stainless steel surface by covalent immobilization of enzymes. Physical characterization and interfacial enzymatic activity. 2056 1


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