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)

A dynamical model of interdomain "hinge bending" of T4 lysozyme in aqueous solution has been developed on the basis of molecular dynamics (MD) simulation. The MD model study provides a description of the conformational reorganization expected to occur for the protein in aqueous solution as compared to the crystalline environment. Three different 500 ps molecular dynamics simulations were calculated, each using a distinctly different crystal conformation of T4 lysozyme as the starting points of the MD simulations. Crystal structures of wild-type lysozyme and "open" and "closed" forms of M61 variant structures were analyzed in this study. Large-scale, molecular-conformational rearrangements were observed in all three simulations, and the largest structural change was found for the open form of the M61 allomorph. All three simulated proteins had closed relative to the wild-type crystal structure, and the closure of the "jaws" of the active site cleft occurred gradually over the time course of the trajectories. The time average MD structures, calculated over the final 50 ps of each trajectory, had all adapted to conformations more similar to each other than to their incipient crystal forms. Using a similar MD protocol on cytochrome P450BM-3 [M. D. Paulsen and R. L. Ornstein (1995) Proteins: Structure Function and Genetics, Vol. 27, pp. 237-243] we have found that the opposite type of motion relative to the starting crystal structure, that is, the open form of the crystal structure, had opened to a greater degree relative to the incipient crystal structure form. Therefore we do not believe that either result is merely a simulation artifact, but rather the protein dynamics are due to protein relaxation in the absence of crystal packing forces in the simulated solution environments.
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PMID:Protein hinge bending as seen in molecular dynamics simulations of native and M61 mutant T4 lysozymes. 909 76

Amine fluoride (AmF)- and stannous fluoride (SnF2)-containing products were found to have a therapeutic effect on gingivitis and periodontitis. This effect was suggested to correlate with the antibacterial activity of the fluoride compounds. However, their effect on inflammatory cell function can also play a role in the therapeutic effect on gingival inflammation. The present study was designed to test the effects of AmF, SnF2, and an AmF/SnF2 combination on the function of human peripheral blood neutrophils, as compared with effects of chlorhexidine and salicylic acid. Neutrophils were isolated from human blood by ficoll centrifugation followed by dextran sedimentation. The neutrophils were pre-incubated with AmF, SnF2, or AmF/SnF2, followed by stimulation with fMLP. Cell vitality was verified by trypan-blue exclusion (> 95% vitality at all tested concentrations). Superoxide production was measured by cytochrome C reduction and the enzymatic activity of lysozyme and beta-glucoronidase by optical density measurement of substrate conversion. The results showed that AmF, SnF2, or AmF/SnF2 enhanced by two- to three-fold the superoxide release from fMLP-stimulated human neutrophils. Furthermore, the effective concentration of the AmF/SnF2 combination was several-fold lower than that of AmF or SnF2 alone (10 nM for AmF, 0.5 microM for SnF2, and 3 pM for SnF2/AmF). On the other hand, chlorhexidine and salicylic acid were found to reduce superoxide production by the cells. All the tested compounds had no effect on granular enzyme release by the stimulated neutrophils. The results suggest that AmF and SnF2 enhance the oxygen-dependent antibacterial activity of neutrophils. This effect may contribute to a more efficient elimination of bacteria from the periodontal environment, resulting in improvement in gingival health.
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PMID:Effect of amine and stannous fluoride on human neutrophil functions in vitro. 920 71

Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N epsilon-ubiquitinyl-cytochrome-c, N epsilon-K48polyubiquitinyl-lysozyme, or an N alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.
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PMID:Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. 952 56

Alcohols have been shown to cause a conformational transition of proteins into a new stable conformational state resembling that of the "molten globule intermediate" characterized by high alpha-helical content and disrupted tertiary structure. We have studied the effect of monohydric alcohols on the stability and structural characteristics of small globular protein hen egg white lysozyme by the combined use of differential scanning calorimetry, circular dichroism, and nuclear magnetic resonance spectroscopy. The protein stability was found to be significantly decreased with increasing alcohol concentration, and, in presence of moderate to higher alcohol concentrations, depending on the pH and alcohol studied, the protein was found to be unfolded even at 4 degrees C. Correlation between thermal stability and alpha-helicity of several small globular proteins like hen egg white lysozyme, horse heart cytochrome C, and bovine carbonic anhydrase B, observed in presence of increasing alcohol concentrations, suggests that probably alcohols induce helical structures in unfolded protein. The temperature-dependent near- and far-UV circular dichroism and proton nuclear magnetic resonance spectroscopic studies on lysozyme in the presence of 2,2,2-trifluoroethanol and methanol, respectively, showed that alcohols do induce significantly higher helical structures in unfolded protein compared to folded protein. The results presented in this paper suggest that the molten globule intermediate of proteins in the presence of high alcohols as reported earlier is due to alcohol-induced local folding rather than global folding of unfolded protein and hence is an off-pathway product and not a real folding intermediate.
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PMID:Alcohol-induced molten globule intermediates of proteins: are they real folding intermediates or off pathway products? 973 68

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.
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PMID:Protein stability: functional dependence of denaturational Gibbs energy on urea concentration. 1002 41

This paper describes the modification of the method by Coon and Pernecky (Meth. Enzymol. 1996, 272, 25-34) for purification of truncated (delta 2-27) recombinant form of cytochrome P450 2B4 expressed in E. coli as fusion protein with glutathione-S-transferase. The modifications included optimisation of conditions for proteolytic reaction of fusion protein with thrombine, removal of this protease from purified cytochrome P450 preparations using column chromatography on hydroxyapatite, introduction of the additional step for obtaining of spheroplasts using of lysozyme, and optimisation of conditions for enzyme stabilisation during of its purification and storage. The overall yield of purified cytochrome was 20% and the specific content of P450 was 14,5 nmol/mg protein was measured. This method is suitable for large-scale isolation of high purified cytochromes P450 which are necessary for study of structure-functional relationships of this hemoprotein with protein partners as well as for investigation of its structure and mechanism of action.
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PMID:[Isolation and purification of recombinant truncated (delta2-27) cytochrome P450 2B4 expressed in E. coli cells as a fusion protein with glutathione-S-transferase]. 1020 25

To investigate the effects of musk-1, a glucoprotein component isolated from the water extract of musk, on the release of superoxide anion as well as beta-glucuronidase and lysozyme of rat polymorphonuclear leukocytes activated by fMLP. An in vitro incubation system with rat polymorphonuclear leukocytes was used. Superoxide anion production was determined by cytochrome C reduction. beta-glucuronidase and lysozyme release was quantitated by enzyme reactions in which phenolphthaleinglucuronic acid and micrococcus lysodeikticus were as the substrates, respectively. In comparison with control, musk-1 at final concentrations of 1-100 micrograms/ml can increase superoxide anion production by 23.0%-83.6% and decrease beta-glucuronidase and lysozyme release by 7%-47% and 9%-22%, respectively, in rat polymorphonuclear leukocytes. It is concluded that Musk-1 can significantly affect the functions of rat polymorphonuclear leukocytes. Therefore, inhibition of lysosomal enzyme release might be considered as one of the mechanisms of anti-inflammatory role of musk.
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PMID:[Effects of the glucoprotein component of musk on the functions of rat polymorphonuclear leukocytes activated by fMLP in vitro]. 1045 95

Although the conformational changes accompanying the oxidation of ferrocytochrome c by the transfer of an electron to cytochrome a are small, they may contribute to the regulation of the electron transfer by transient storage of the liberated energy as strain and atomic vibrations. Both the electron transfer and the conformational changes seem to be controlled by an attractor, i.e. by a manifestation of a deterministic chaos. The putative attractor is regular and is, for the reaction involving the inner monomer of ferricytochrome c (I), of the order of 3.03 +/- 0.03. The conformational changes involving the outer monomer of ferricytochrome c (O) seem also to be controlled by a regular attractor, but its order is 4.2 +/- 0.2. The low order of the coupled reactions of electron transfer and conformational change suggests that it is essential to the electron transfer process in the respiratory chain. Since the order of attractors of other proteins correlates with the vectorial description of the function (1.0 for myoglobin, 2.0 for chymotrypsin and lysozyme, 3.0 for an abenzyme), the value for cyt. c indicates that not only the electron transfer, but also an additional reaction, e.g. the conformational change, are essential for the function of this protein. Hence, the study of protein attractors may yield information on important details, which could not be obtained by other methods.
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PMID:Attractor control of the redox reactions of bovine cytochrome c. 1069 Nov 90

At present there is considerable interest in labeling peptides with Tc-99m for the development of target specific radiopharmaceuticals for imaging purposes. In the present study the conjugation of the bifunctional coupling agent succinimidyl-hydrazinonicotinamide (S-HYNIC) was studied and optimized in a series of peptides [molecular weight (MW) 6.5-14.3 kDa]. Aprotinin (MW 6.5 kDa), cytochrome C (MW 12.4 kDa), alpha-lactalbumin (MW 14.2 kDa), and lysozyme (MW 14.3 kDa) were conjugated with S-HYNIC via the epsilon amino groups of their lysine residues. The effects of molar conjugation ratio, reaction temperature, pH, and protein concentration were studied. Reaction products were analyzed both with respect to the HYNIC-substitution ratio (spectrophotometrically) as well as to the labeling efficiency silica gel-instant thin layer chromatography (SG-ITLC) and molecular size fast performance liquid chromatography (FPLC). The effects of conjugation on biological activity were studied in three proteins binding to receptors on leukocytes: interleukin-8 (MW 8.5 kDa), interleukin-1alpha (MW 17 kDa), and interleukin-1 receptor antagonist (MW 17 kDa). The labeling efficiency of aprotinin, cytochrome c, alpha-lactalbumin, and lysozyme conjugated under optimal conjugation conditions exceeded 90%. Specific activities obtained were up to 7.5 MBq/microg. Conjugation was most efficient at 0 degrees C (as compared to 20 and 40 degrees C), at pH 8.2 (as compared to 6.0, 7.2, and 9.5), and at protein concentrations > or = 2. 5 mg/mL. In general, efficiency increased with increasing molar conjugation ratio (protein-HYNIC-ratio 1:3 < 1:6 < 1:15<1:30). For the receptor binding proteins, biological activity was preserved only under the mildest conjugation conditions. For each of these proteins an inverse relation between labeling efficiency and receptor binding capacity was found. Labeling proteins with (99m)Tc using S-HYNIC is easy, rapid, and efficient, and preparations with high specific activity can be obtained. However, biological activity of proteins may be lost at high HYNIC-substitution ratios. With the proteins tested here a careful balancing of reaction conditions resulted in acceptable, although suboptimal, labeling efficiencies and preservation of biological activity.
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PMID:Labeling proteins with Tc-99m via hydrazinonicotinamide (HYNIC): optimization of the conjugation reaction. 1105 76

The adsorption of lysozyme and cytochrome C on phosphatidylcholine liposomes essentially changes the physical properties of the phospholipid membranes and under certain circumstances greatly affects the stability of the colloid dispersion by inducing bridging liposome flocculation. This study was designed to examine experimentally the influence of liposome size on two kinetic parameters of the flocculation, its rate constant and activation energy. As the liposome radius increased in the range 50-500 nm, the activation energy tended to decrease, resulting in an increased flocculation rate, except for the flocculation of 400-nm liposomes, which was greatly impeded. The pronounced influence of the liposome size on the flocculation rate constant was evident, since a well-defined minimum in the kinetic rate of flocculation of 400-nm liposomes was detected experimentally. The obtained nonlinear radius dependencies of the flocculation rates and activation energies are interpreted in terms of the bridging mechanism of the protein-induced liposome flocculation and the supplementary concept of the stability of thin liquid films formed between approaching protein-adsorbed liposomes. Copyright 2000 Academic Press.
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PMID:Size Dependence of Protein-Induced Flocculation of Phosphatidylcholine Liposomes. 1140 44


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