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 potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 A. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 A. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).
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PMID:A new additive for protein crystallization. 795 78

We have measured the metabolic stabilities of wild-type and 17 temperature-sensitive mutants of T4 lysozyme in HeLa cells, in Xenopus egg extract, and in reticulocyte lysate. [35S]Methionine-labeled T4 lysozymes were expressed in Escherichia coli, purified, injected into HeLa cells, and their degradation rates were determined. Wild-type T4 lysozyme has a half-life of 4 h; the half-lives of 16 lysozyme variants ranged from 2 to 10 h. Surprisingly, the most temperature-sensitive enzyme in the set, R96H, was significantly more stable (half-life = 10 h). Different T4 lysozyme variants yield conflicting answers to the proposed relationship between thermal and metabolic stabilities. For mutations at Thr157 there is no correlation between melting temperature and half-life. By contrast, T4 lysozymes mutated at various positions show a definite correlation between the two parameters. Treatment of injected HeLa cells with the lysosomotropic agents chloroquine or ammonium chloride did not alter the stability of T4 lysozyme. However, the enzyme's half-life increased 10-fold in HeLa cells depleted of ATP. Although T4 lysozyme is degraded rapidly within HeLa cells, the molecule is stable in reticulocyte lysate and Xenopus egg extract. Presumably, there is a specific proteolytic event(s) in HeLa cells which is not manifest in the in vitro extracts.
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PMID:On the relationship between the metabolic and thermodynamic stabilities of T4 lysozymes. Measurements in eukaryotic cells. 796 93

Two mutants of human lysozyme were synthesized. Mutant A92D, in which Ala92 was substituted by Asp, contains a partial Ca(2+)-binding site and mutant M4, in which Ala83, Gln86, Asn88 and Ala92 were replaced by Lys, Asp, Asp and Asp respectively, contains the complete Ca(2+)-binding site of bovine alpha-lactalbumin. The Ca(2+)-binding constants of wild type human lysozyme and of mutants A92D and M4, measured at 25 degrees C and pH 7.5, were 2(+/- 1) x 10(2) M-1, 8(+/- 2) x 10(3) M-1 and 9(+/- 0.5) x 10(6) M-1 respectively. Information gathered from microcalorimetric and CD spectroscopic measurements indicates that the conformational changes of the M4 mutant lysozyme, induced by Ca2+ binding, are smaller than those observed for bovine alpha-lactalbumin and for the Ca(2+)-binding equine lysozyme. At pH 4.5, the thermostability of both the apo and Ca2+ forms of the A92D human was decreased in comparison with that of native human lysozyme. In particular, within the apo form of this mutant an alpha-helix-containing sequence was destabilized. In contrast, at the same pH the thermostability of the apo and Ca2+ forms of the M4 mutant lysozyme was increased. The epsilon-ammonium group of the Lys83 side chain is assumed to be responsible for the stabilization of the apo form of this mutant.
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PMID:Stability effects associated with the introduction of a partial and a complete Ca(2+)-binding site into human lysozyme. 823 35

Monitoring of microbial DNA in soils by dot blot hybridization and PCR analysis is a useful technique for gaining insight into the survival and impact of genetically modified micro-organisms released in the environment. Most methods of DNA isolation from soils require a large number of purification steps rendering them unsuitable for quantitative analysis of multiple samples. Here we describe a very rapid method for the isolation and purification of multiple samples of soil DNA that can be used directly for dot blot hybridization and PCR analysis. Soil DNA extracts are prepared by lysozyme/SDS treatment at pH 9.0 and purified by ammonium acetate precipitation and Sephadex G50 gel filtration. In a practical application of this method, sandy soil samples were seeded with Alcaligenes eutrophus cells and exposed to high temperature (42 degrees C) or desiccation. As a result, the number of culturable A. eutrophus cells which could be recovered from the soil samples quickly declined. However, the concentration of a marker gene encoding resistance to cadmium, cobalt and zinc (czc) remained unaltered.
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PMID:Rapid method for purification of soil DNA for hybridization and PCR analysis. 826 Nov 67

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

The specificity of interactions between biological macromolecules and their ligands may be partially attributed to the directional properties of hydrogen bonds. We have now extended the GRID method (Goodford, P. J. J. Med. Chem. 1985, 28, 849. Boobbyer, D. N. A.; Goodford, P. J.; McWhinnie, P. M.; Wade, R. C. J. Med. Chem. 1989, 32, 1083), of determining energetically favorable ligand binding sites on molecules of known structure, in order to improve the treatment of groups which can make multiple hydrogen bonds. In this method, the interaction energy between a probe (a small chemical group that may be part of a larger ligand) and a target molecule is calculated using an energy function which includes a hydrogen bond term which is dependent on the length of the hydrogen bond, its orientation at the hydrogen-bonding atoms, and their chemical character. The methods described in the preceding paper (Wade, R. C.; Clark, K. J.; Goodford, P. J. J. Med. Chem., preceding paper in this issue) for probes capable of making two hydrogen bonds are here extended to the following probes which have the ability to make more than two hydrogen bonds: ammonium-NH3+, amine-NH2, sp3-hybridized hydroxyl, and water. Use of the improved GRID procedure is demonstrated by the determination of the conformation of an amino acid side chain at the subunit interface in hemoglobin and of the location of water binding sites in human lysozyme.
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PMID:Further development of hydrogen bond functions for use in determining energetically favorable binding sites on molecules of known structure. 2. Ligand probe groups with the ability to form more than two hydrogen bonds. 842 Dec 81

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.
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PMID:Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin. 852 15

The effects of various anions on decreasing the solubility of acidic Hypoderma lineatum collagenase at pH 7.2 and 18 degrees C were qualitatively defined by replacing the crystallizing agent of known crystallization conditions by various ammonium salts. The solubility curves measured in the presence of the sulfate, phosphate, citrate, and chloride ammonium salts gave the following ranking of anions: HPO4(2-)/H2PO4- > SO4(2-) > citrate 3-/citrate2- >> Cl-. This order is in agreement with the Hofmeister series. In a previous study on the solubility at pH 4.5 of lysozyme, a basic protein, the effectiveness of anions in decreasing the solubility was found to be in the reverse order. This suggests that the effectiveness of anions in the crystallization of proteins is dependent on the net charge of the protein, i.e., depending on whether a basic protein is crystallized at acidic pH or an acidic protein at basic pH.
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PMID:Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2. 853 49

A new method for the isolation of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using electroelution with a modified buffer system, is described. The method is suitable for direct characterization by electrospray-ionization mass spectrometry (ESI-MS), or alternatively matrix-assisted laser desorption-ionization (MALDI), no additional purification steps being required. Following separation by SDS-PAGE, proteins were stained with Coomassie Blue, and isolated by electroelution using SDS-free ammonium acetate (pH 2.5) as elution buffer. The polarity of the electroelution system was reversed, which, upon in situ dissociation of protein-SDS complexes, resulted in migration of free proteins to the cathode under the conditions employed. Recovery rates of 25-58% were determined for model proteins. Analyses by ESI-MS provided exact molecular weight determinations of isolated proteins and of protein mixtures not resolved by SDS-PAGE. Essentially SDS-free molecular ions were obtained, except for bovine serum albumin with one SDS-adduct. Charge distribution of molecular ions were similar to those of the native proteins. The effects of beta-mercaptoethanol (beta-ME) and dithiothreitol (DTT) during electrophoresis were studied with hen egg white lysozyme, revealing the formation of mixed disulfide adducts between proteins and reducing agents. In a first bioanalytical application, hemofiltrate proteins from a patient with uremia were separated by SDS-PAGE. An ESI-MS analysis of the proteins isolated from the two most intensive gel bands enabled exact molecular weight determinations, and demonstrated that a gel band of ca. 17 kDa consisted of two different proteins.
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PMID:Direct isolation of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by electrospray-ionization mass spectrometry. 878 11

Reduced lysozyme was renatured by sulfhydryl-disulfide interchange reactions at pH 8.0 in the presence of 4 M urea, with or without additives at 40 degrees C. In the absence of additives, the final folding yield of reduced lysozyme was approximately 40%. In the presence of sarcosine, glycerol, ammonium sulfate, N-acetyl glucosamine and glucose, its folding yields increased in all cases. In particular, yields increased up to 90% in the presence of 4 M sarcosine. On the other hand, the melting temperatures of lysozyme with or without additives in 0.02 M citrate buffer (pH 6.0) were evaluated using differential scanning calorimetry. In the absence of additive, the melting temperature of lysozyme was 73.8 degrees C. In the presence of additives, all melting temperatures were higher than that of lysozyme in the absence of additives. Moreover, there was a good correlation on addition of additives between an increase in the folding yield of reduced lysozyme with 4 M urea and an increase in the melting temperature without 4 M urea. Therefore, we conclude that additives, which stabilize native lysozyme, are effective at increasing the folding yield of reduced lysozyme in 4 M urea.
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PMID:Effect of additives on the renaturation of reduced lysozyme in the presence of 4 M urea. 879 46


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