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)

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins--thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2+)--allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii) when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).
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PMID:Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. 253 16

The three-dimensional structure of a protein is governed by the thermodynamical principle established experimentally by Anfinsen, Isemura, and others. The rapidity of the folding process is another important key phenomenon. With these basics in mind an island model is proposed which requires a restricted folding pathway. A physicochemical method of the prediction of alpha-helices and beta-strands is also discussed. By virtue of the long-range hydrophobic interaction and the specific interactions between hydrophobic residues which are determined by the basic idea underlying the island model, one can fold the polypeptide chain into a tertiary structure upon determination of the secondary structures. Several examples of folding are presented. In myoglobin the heme group must be considered to reach the correct final tertiary structure. In lysozyme and phospholipase, the disulfide bondings are necessary to fasten the polypeptide chain. The selection of proper cysteine pairs among other possible ones is carried out by drawing the lampshades (locus of H atom of SH) of cysteines. In flavodoxin and thioredoxin the formation of parallel beta-structure from beta-strands is considered. The formations of antiparallel beta-structure in lysozyme and phospholipase are also discussed.
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PMID:Principles of protein architecture. 269 41

The chaperone DnaK can be released (up to 40%) by osmotic shock, a procedure which is known to release the periplasmic proteins and a select group of cytoplasmic proteins (including thioredoxin and elongation factor Tu) possibly associated with the inner face of the inner membrane. As distinct from periplasmic proteins, DnaK is retained within spheroplasts prepared with lysozyme and EDTA. The ability to isolate DnaK with a membrane fraction prepared under gentle lysis conditions supports a peripheral association between DnaK and the cytoplasmic membrane. Furthermore, heat shock transiently increases the localization of DnaK in the osmotic-shock-sensitive compartment of the cytoplasm. We conclude that DnaK belongs to the select group of cytoplasmic proteins released by osmotic shock, which are possibly located at Bayer adhesion sites, where the inner and outer membranes are contiguous.
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PMID:Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm. 796 73

The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+ sequestration or as "molecular chaperones" in the folding and assembly of membrane or secreted proteins. Based on the ability of many chaperones to bind selectively to unfolded proteins and to dissociate from them upon ATP hydrolysis, we developed an affinity chromatography method to isolate proteins with these characteristics from pancreatic or liver ER. Seven ER proteins bound selectively to denatured protein columns and were specifically eluted by ATP (10(-6) M) but not by a nonhydrolyzable ATP analog. These proteins were identified with antibodies and microsequencing as the ER chaperone BiP (grp78), grp94, calreticulin, a novel 46-kDa protein that binds azido-ATP, as well as three members of the thioredoxin superfamily: protein-disulfide isomerase, ERp72, and a previously reported 50-kDa protein (p50). This set of seven proteins bound to and was eluted with ATP from a variety of denatured proteins, including histone, gelatin, alpha fetoprotein, thyroglobulin, lysozyme, casein, and IgG. The release of grp94, protein-disulfide isomerase, ERp72, calreticulin, and p50 was stimulated by Ca2+ in the presence of ATP. These proteins thus appear to function as Ca(2+)-dependent chaperones, which may account for the Ca2+ and ATP requirement for protein folding in the ER.
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PMID:A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca(2+)-binding proteins and members of the thioredoxin superfamily. 829 23

Protein disulfide isomerase has broad specificity in the catalysis of the formation and rearrangement of native disulfide bonds in proteins. This enzyme has two independent thioredoxin-like active sites (-CGHC-) and a peptide binding site. However, the mechanisms involving the catalytic processes are not clearly understood. It was reported that the enzyme associates with scrambled pancreatic ribonuclease A in vitro, and with misfolded human lysozyme in vivo. In the present study, recombinant human interleukin 2 has been chosen to probe the reaction intermediate in the reaction with the enzyme. We have identified and characterized a covalent associate formed in vitro by SDS-PAGE and Western blot analysis. This associate has a molecular weight of 71-72 kDa, the approximate sum of the molecular weights of the enzyme and the substrate. Western blot analysis confirmed that it formed via an intermolecular disulfide bond. Upon treatment with 2-mercaptoethanol, this bond was cleaved.
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PMID:Covalent association of protein disulfide isomerase with recombinant human interleukin 2 in vitro. 866 Mar 62

Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenbaugh, M. J. (1996) Protein Expression Purif. 7, 281-288). PDI is known to include catalytic active sites in two separate thioredoxin-like domains, one near the amino terminus and another near the carboxyl terminus. To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with cysteine to serine mutations at the first cysteine of one or both of the CGHC active site sequences. Trichoplusia ni insect cells were coinfected with a baculovirus vector coding for IgG in concert with either the wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl-terminal mutant (PDI-C), or mutant with both active sites altered (PDI-NC). Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells. To evaluate the effect of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with 35S-amino-acids for different periods, and the soluble immunoglobulins were immunoprecipitated from clarified cell lysates and culture medium using anti-IgG antibodies. Only coinfections with the wild-type PDI and PDI-N mutant led to increased immunoglobulin solubility and higher IgG secretion. In contrast, infection with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus. Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI protein were detected in soluble form. The capacity for PDI-N to increase immunoglobulin solubility whereas the PDI-C mutant lowered solubility indicates that the amino- and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the active site. Furthermore, mutations at the active site of the carboxyl-terminal thioredoxin domain result in PDI variants that can act as anti-chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).
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PMID:Thioredoxin domain non-equivalence and anti-chaperone activity of protein disulfide isomerase mutants in vivo. 927 9

During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92. To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92. These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies. For the preparation of lysozyme samples, a refolding system using thioredoxin was established. This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days. The calcium ion dependence of the melting temperature (Tm) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5. The Tm values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration. However, the Tm of HLQ86D/D91Q/A92D was 4 degrees higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(+/-0.25)x10(2) M(-1) at pH 4.5. Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography. These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability. The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed.
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PMID:Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies. 974 21

When introduced into a chemically defined minimal medium supplemented with 1 mM sodium selenite (79 ppm Se(o)), Bacillus subtilis was found to undergo a series of morphological and biochemical adaptations. The morphological changes included the formation of "round bodies" associated with the detoxification of selenite to elemental selenium. Round bodies observed transiently were not apparent during balanced growth of cells adapted previously to selenite-containing medium. Under balanced growth conditions, cell structures similar to "round bodies", could be produced by treating cells with lysozyme. The selenite-induced structural alterations in cells were accompanied by an increase in the content of thioredoxin and the associated enzyme, NADP-thioredoxin reductase. The results suggest that the biovalence transformation of high levels of selenite may involve a dithiol system.
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PMID:Morphological and biochemical responses of Bacillus subtilis to selenite stress. 1061 98

A site-specific and efficient fluorolabeling of antibody variable regions with green fluorescent protein (GFP) variants and its application to an energy transfer-based homogeneous fluoroimmunoassay (open sandwich FIA) were attempted. Two chimeric proteins, Trx-V(H)-EBFP and Trx-V(L)-EGFP, consisting of V(H) and V(L) fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFP color variants, EBFP and EGFP, respectively, were designed to be expressed in cytoplasm of trxB - mutant Escherichia coli as fusions with thioredoxin from E.coli The mixture of two proteins could be purified with HEL-affinity chromatography, retaining sufficient intrinsic fluorescence and binding activity to HEL. A significant increase in fluorescence resonance energy transfer (FRET) dependent on HEL concentration was observed, indicating the reassociation of the V(H) and V(L) domains of these chimeric proteins due to co-existing antigen. With this open sandwich FIA, an HEL concentration of 1-100 microg/ml could be non-competitively determined. The assay could be performed in a microplate format and took only a few minutes to obtain a sufficient signal after simple mixing of the chimeric proteins with samples. This represents the first demonstration that the FRET between GFP variants is applicable to homogeneous immunoassay.
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PMID:Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay. 1083 11

We have studied the one-electron reduction of oxidized Chlamydomonas reinhardtii thioredoxin and compared it to that of hen egg white lysozyme, using CO(2)(*) (-) free radicals as reductants. This comparison shows that the thioredoxin disulfide/thiol redox couple has different properties than that of lysozyme: the disulfide radical pK(a) is much lower (around 5 for small disulfides, 4.62 for lysozyme, <3 for thioredoxin). To get a better understanding of the modulation of the thioredoxin redox properties we have constructed the mutants W35A and D30A. Their reduction by pulse radiolysis indicates that W35 strongly controls both the disulfide radical acidity (the pK(a) in W35A is equal to ca. 4), and the thiol reactivity. Asp30 is also involved in the control of proton transfer to the disulfide free radical. In addition, its removal seems to increase the reduction potential of the thioredoxin thiyl/thiol couple. Overall, the reduction properties of thioredoxin confirm its nature as a unique reductant.
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PMID:Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme: a pulse radiolysis study. 1092 22


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