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)

The amino acid sequence of copper pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 20, 77, and 113 for Lady Amherst's pheasant lysozyme and seven amino acid substitutions at positions 3, 15, 20, 41, 113, 121, and 124 for hen lysozyme. Phenylalanine at position 20 was newly detected in avian lysozyme.
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PMID:The amino acid sequence of copper pheasant lysozyme. 776 70

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

A new procedure has been developed to immobilize iminodiacetic acid (IDA) onto the surface of silica supports, such as LiChrospher Si-1000 and 1.5-microns nonporous silica, for use in high-performance immobilized metal affinity chromatography (HPIMAC) of proteins. This IDA immobilization method has been achieved through the synthesis of a new silylation reagent, 1-(iminodiacetic acid di-tert-butylester)-3-glycidoxy-propyltrimethoxysilane (IDA-silane). Various modified silicas of different ligand densities have been prepared by using mixtures between 1 and 100% of the IDA-silane diluted with the corresponding 3-glycidoxy-propyltrimethoxysilane (GLYMO-silane). Frontal analysis was used with the IDA-Cu(II)-Concanavalin-A and IDA-Cu(II)-lysozyme systems to evaluate the capacity and the association constants for these HPIMAC sorbents. With these metal chelate sorbents the specific binding capacity per unit area increased continuously with the ligand density for the nonporous sorbents but reached a maximum at about 50% of the maximum ligand coverage for the porous sorbents. The association constant for the chelate-protein complex was highest for both concanavalin-A (Con-A) and lysozyme (HEWL) at the highest ligand density and decreased with lower ligand density. These observations have been evaluated in terms of the accessibility of histidine residues on the surface of the two test proteins and their ability to act as binding sites for the copper ions in the coordination complex. The experimental data indicate that both steric and conformational effects result in multiple classes of binding phenomena with Con-A and HEWL at high ligand concentrations. These experimental results provide a useful guideline for the design of silica-based sorbents for application in the HPIMAC of proteins.
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PMID:Influence of ligand density on the properties of metal-chelate affinity supports. 838 18

Conditions for the immobilization of O-phosphoserine (OPS) to epoxy-activated Sepharose CL-4B are described. The binding behaviour of OPS and iminodiacetic acid (IDA) immobilized onto Sepharose CL-4B, toward the hard Lewis metal ions Al3+, Fe3+, Ca2+ and Yb3+, and Cu2+ ion as a borderline metal ion control, over the pH range pH 4.0 to pH 8.0, was examined. Immobilized OPS shows a stronger affinity for Fe3+ and Al3+ ions but a lower affinity for Cu2+ and Yb3+ ions, compared to immobilized iminodiacetic acid (IDA), over the equilibrating range examined. Immobilized OPS-Mn+ was screened for protein binding using as model proteins tuna heart cytochrome c (THCC), horse myoglobin (HMYO) and hen egg while lysozyme (HEWL) over the pH range 5.5 to 8.0. Immobilized OPS-Fe3+ bound THCC under all the examined equilibrating conditions, bound HMYO between pH 5.5 and pH 7.0 and did not bind HEWL under any condition examined. Immobilized OPS thus presents an additional mode of metal ion and protein selectivity in immobilized-metal affinity chromatography.
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PMID:High-performance liquid chromatography of amino acids, peptides and proteins. CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins. 840 20

To evaluate their usefulness as chemical indicators of cumulative oxidative damage to proteins, we studied the kinetics and extent of formation of ortho-tyrosine (o-Tyr), dityrosine (DT), and dityrosine-like fluorescence (Ex = 317 nm, Em = 407 nm) in the model proteins RNase and lysozyme exposed to radiolytic and metal-catalyzed (H2O2/Cu2+) oxidation (MCO). Although there were protein-dependent differences, o-Tyr, DT, and fluorescence increased coordinately during oxidation of the proteins in both oxidation systems. The contribution of DT to total dityrosine-like fluorescence in oxidized proteins varied from 2-100%, depending on the protein, type of oxidation, and extent of oxidative damage. In proteins exposed to MCO, DT typically accounted for > 50% of the fluorescence at DT wavelengths. These studies indicate that o-Tyr and DT should be useful chemical markers of cumulative exposure of proteins to MCO in vitro and in vivo.
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PMID:Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation. 850 73

The interactions of horse muscle myoglobin (MYO), tuna heart cytochrome c (CYT), and hen egg white lysozyme (LYS) with three different immobilized metal ion affinity (IMAC) adsorbents involving the chelated complexes of the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline Lewis metal ion Cu2+ have been investigated in the presence of low- and high-ionic strength buffers and at two different pH values. In contrast to the selectivity behavior noted with buffers of high ionic strength, with low-ionic strength buffers, these three proteins interact with the hard metal ion IMAC adsorbents in a manner more characteristic of cation exchange behavior, although in contrast to the cation exchange chromatography of these proteins, as the pH value of the elution buffer was increased, the retention also increased. The selectivity differences observed under these conditions appear to be due to the formation of hydrolytic complexes of these immobilized metal ion chelate systems involving a change in the coordination geometry of the im-M(n+)-chelate at higher pH values. The experimental observations have been evaluated in terms of the effective charge on the immobilized metal ion chelate complex and the charge characteristics of the specific proteins.
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PMID:Application of immobilized metal ion chelate complexes as pseudocation exchange adsorbents for protein separation. 855 75

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline metal ion Cu2+, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the range pH 5.5-8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized Mn+-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe3+-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe3+-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metal-ion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surface-exposed aspartic and glutamic acid residues on the protein with the immobilized Mn+-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu2+-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number of clustering of the surface-accessible aspartic and glutamic residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.
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PMID:Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues. 859 82

The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.
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PMID:A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme. 861 88

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.
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PMID:Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli. 861 89

Periplasmic expression of recombinant proteins presents many potential benefits that may aid recovery of the protein product. Muramidases are the preferred agents in effecting selective release of recombinant proteins from the periplasm of E. coli and other Gram negative bacteria. Unfortunately cost restricts the use of pure lytic enzymes at large-scale and their removal as process contaminants adds to later purification demands. We constructed a reusable version of bacteriophage T4 lysozyme, by fusing a His-Gln-(His)3 peptide sequence to the C-terminus of a cysteine-free pseudo wild type bacteriophage T4 lysozyme. The peptide tail allowed rapid and high-level recovery on IDA Sepharose columns charged with Zn2+, Ni2+ and Cu2+ ions. The binding to metal-charged supports was specifically mediated by the histidine-rich tail as no binding was observed for the original cysteine-free pseudo wild type lysozyme. The strength of retention of polyhistidine recombinant T4 lysozyme on charged supports followed the expected Cu > Ni > Zn pattern, but there were few differences in the levels of purity and recovery of the modified enzyme, from columns charged with the different metal ions.
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PMID:Expression and purification of a recombinant metal-binding T4 lysozyme fusion protein. 887 73


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