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)

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.
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PMID:Modification of arginine and lysine in proteins with 2,4-pentanedione. 0 43

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.
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PMID:Phage T4 lysozyme. Physical properties and reversible unfolding. 23 26

Guanidine hydrochloride (GdnHCl) and urea denaturations of lysozyme have been observed at various temperatures by measuring changes in fluorescence. Both transitions appear to be two state, with GdnHCl almost twice as effecitve a denaturant as urea for this protein. By plotting the denaturant concentrations at midpoint of the transition vs. the experimental temperature, it can be demonstrated that urea-denatured lysozyme does not obtain the degree of unfolding found in lysozyme denatured by GdnHCl.
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PMID:Comparison of temperature effect on the guanidine hydrochloride and urea denaturations of lysozyme. 53 98

Wild-type T4 lysozyme contains unpaired cysteine residues at positions 54 and 97. To investigate the role these residues play in the thermal inactivation of the wild-type, we constructed a double mutant with these cysteines replaced with valine and serine. This molecule, T4 lysozyme (C54V/C97S), is more stable than the wild-type to inactivation at 70 degrees C at pH 6.5 and 8.0. Guanidine hydrochloride reactivation experiments and SDS-PAGE on the inactivated products show that the wild-type is susceptible to varying degrees of oxidative damage, depending on buffer conditions, while the cysteine-minus mutant inactivates only by other pathways. The products of thermal, oxidative inactivation of the wild-type are disulfide-linked oligomers. The dependence of inactivation rate on temperature suggests that the formation of these aggregates depends on prior thermal unfolding of the T4 lysozyme molecule.
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PMID:The role of cysteine oxidation in the thermal inactivation of T4 lysozyme. 350 92

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.
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PMID:Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme. 1072 42

Large scale abstraction and isolation of bacterially synthesized, recombinant-DNA-derived, porcine growth hormone (r-pST) is described. The r-pGH is found in genetic engineering E. coli as the form of inclusion bodies. Pellet fraction which were mainly inclusion bodies, after cell breakage and centrifugation, were collected. Cell envelope components, such as protein, lipid, endotoxin and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. Inclusion bodies were dissolved using 6 mol/L guanidine/HCl and air oxidation is then carried out in the presence of the guanidine/HCl. The Guanidine/HCl protein mixture were diluted by renaturation solution. Guanidine/HCl were removed by dialysis and then correctly refolded, oxidized r-pGH were obtained. Injection experiment of hypophysectomized rats proved r-pST with high native bioactivity was obtained.
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PMID:[Isolation, purification and renaturation of recombinant-DNA-derived porcine somatotropin]. 1191 Jul 69

A general labelling method is presented which allows the determination of the number of guanidine groups (related to arginine and homoarginine in peptides and proteins) by means of mass spectrometry. It implies a guanidine-selective derivatization step with 2,3-butanedione and an arylboronic acid under aqueous, alkaline conditions (pH 8-10). The reaction mixture is then directly analysed by electrospray ionization mass spectrometry without further sample pretreatment. Other amino acids are not affected by this reaction although it is demonstrated that lysine side-chains may be unambiguously identified when they are converted to homoarginine prior to derivatization. Guanidine functionalities, as e.g. in the amino acid arginine, are easily identified by the characteristic mass shift between underivatized and derivatized analyte. The tagging procedure is straightforward and selective for guanidine groups. The influence of several experimental parameters, especially the pH of the solution and the choice of reagents, is examined and the method is applied to various arginine-containing peptides and to lysozyme as a representative protein. Possible applications of this technique and its limitations are discussed.
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PMID:Probing of arginine residues in peptides and proteins using selective tagging and electrospray ionization mass spectrometry. 1293 10

Using 100-fold molar excess of succinic anhydride, about 99% of lysine residues of hen egg white lysozyme (HEWL) were modified. Succinylated (S(99)) HEWL showed both charge and size homogeneity as judged by PAGE and gel filtration, respectively. Hydrodynamic parameters such as Stokes radius and frictional ratio (f/f(o)) showed more expanded conformation of S(99) HEWL compared to native HEWL as evident from the increase in Stokes radius (from 1.36 to 1.86 nm) and f/f(o) (from 0.86 to 1.15) values. Guanidine hydrochloride (GdnHCl) denaturation studies using fluorescence spectroscopy connoted a marked decrease in conformational stability of HEWL upon succinylation. Complete denaturation of S(99) HEWL was achieved at lower GdnHCl concentration ( approximately 3.8 M) compared to native HEWL ( approximately 5 M). Furthermore, free energy of stabilization (DeltaG(D)(H(2)O)) value also showed a notable decrease from 8,559 and 7,956 cal/mol (for native HEWL) to 4,404 and 4,669 cal/mol (for succinylated HEWL) using excitation at 280 and 295 nm, respectively. Both expanded conformation and decreased DeltaG(D)(H(2)O) can be attributed to the increase in the net negative charge on the protein upon succinylation. All these results manifested the importance of positively charged lysine residues in maintaining the conformational stability of HEWL through electrostatic interactions.
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PMID:Succinylation-induced conformational destabilization of lysozyme as studied by guanidine hydrochloride denaturation. 1971 23

Guanidine group (Gnd) functionalized magnetic nanoparticles (Fe3O4@SiO2@NH2-Gnd) were synthesized and characterized in this work for the first time. The characterization of Fe3O4@SiO2@ NH2-Gnd nanoparticles was demonstrated by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectra, vibration sample magnetometer, and zeta potential analyzer. The novel multifunctional nanoparticles were served as a solid-phase extraction sorbent for easy isolation and preconcentration of acidic protein from aqueous solution only using a magnet. Bovine serum albumin (BSA) was selected as a model protein and the main experimental parameters influencing the adsorption and desorption efficiency were investigated and optimized. Under the optimum conditions, the particles reached saturated adsorption within 20 min and exhibited significant specific recognition for the acidic proteins. Fifteen fold enrichment efficiency was achieved and the detection limits was 45 ng x mL(-1) for BSA by capillary electrophoresis (CE). The practical application of the novel nanoparticles as a sorbent for the isolation and preconcentration of acidic proteins from basic proteins was demonstrated by effective separation and enrichment of bovine serum albumin from lysozyme and cytochrome C mixture, which was assayed by CE.
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PMID:Preparation of guanidine group functionalized magnetic nanoparticles and the application in preconcentration and separation of acidic protein. 2240 14

Ile58 of hen egg-white lysozyme (HEL) is buried in the interior of the molecule and is considered to participate in sugar residue binding at subsite C through hydrophobic interaction. The contribution of Ile58 to lysozyme function and stability was investigated by replacement of Ile58 with less hydrophobic residues, Val (I58V) and Ala (I58A). Replacement of Ile58 with Ala decreased substrate binding ability to an N-acetylglucosamine trisaccharide, (GlcNAc)3, and a GlcNAc polymer, chitin, whereas replacement with Val had little effect. Similar results were obtained as to enzymatic activity toward both the bacterial cell substrate and glycol chitin. Kinetic analysis by substrate (GlcNAc)5 revealed that replacement of the Ile residue reduced the sugar residue affinity at subsite C and the rate constant of glycosidic bond cleavage. The rate constant of glycosidic cleavage for mutant I58A was about one-third of that for the wild-type. Guanidine hydrochloride unfolding experiments showed that mutants I58V and I58A were less stable than the wild-type, by 1.88 and 2.88 kcal/mol respectively. Moreover, the stability of the protein inserted at this position decreased linearly with decreasing hydrophobicity of the inserted residue. It appears that the hydrophobicity of Ile58 is an important factor in the efficient substrate binding, enzymatic reaction, and structural stability of HEL.
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PMID:The mutational effect of Ile58 at subsite C in hen egg-white lysozyme on substrate binding, enzymatic activity, and protein stability. 2347 Jul 62


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