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)

Hen egg-white lysozyme (EC 3.2.1.17) was carboxymethylated with iodoacetic acid and the individual products of various degree of modification were isolated by column chromatography on Amberlite CG-50. Peptide mapping of the products obtained reveals that His-15, Lys-1, -33, -96 (or -97) residues are blocked; the Lys-13 and -116 residues are unmodified. Salt activation of the carboxymethylated derivatives is facilitated with the increase of the number of modified groups; this fact is consistent with the increased lytic activity and affinity to substrate of the derivatives at lowered ionic strength.
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PMID:[Preparation and characteristics of carboxymethylated lysozyme derivatives]. 69 4

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.
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PMID:A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein. 103 81

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.
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PMID:Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95. 221 92

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.
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PMID:Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation. 336 12

Exposure of human neutrophils to 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (leukotriene B4, LTB4) resulted in a time- and concentration- (10(-9)-10(-6) M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB4. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB4 prior to contact with cytochalasin B. LTB4-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB4-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) and N-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB4-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive. Neutrophils pretreated with LTB4 or 5(S),12(R),20-trihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB4, an omega-oxidation metabolite of LTB4) were desensitized to the subsequent exposure to LTB4. Cross-desensitization was also demonstrated between LTB4 and 20-OH-LTB4. The stimulus specific nature of LTB4-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB4-pretreated neutrophils. Enzyme release from LTB4-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB4-induced degranulation.
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PMID:Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid. 609 46

Pepstatin A, a chemotactic pentapeptide, elicited a concentration-dependent extracellular release of granule-associated beta-glucuronidase and lysozyme from, and generation of superoxide anion (O2-) by, cytochalasin B (CB)-treated human neutrophils. Prior exposure of neutrophils to pepstatin A before the addition of CB, suppressed, in a time-dependent fashion, the subsequent production of O2- and exocytotic response. The rate and amount of enzymes released and O2- generated by pepstatin A-activated neutrophils were significantly enhanced in the presence of extracellular calcium. Pepstatin A-elicited degranulation and O2- production were suppressed by the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3, 4, 5-trimethoxy) benzoate hydrochloride (TMB-8). Granule exocytosis and O2- generation by pepstatin A-treated neutrophils were suppressed by the sulphydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IA), and by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG). Sodium cyanide was inactive. Preincubation of neutrophils with pepstatin A "desensitized' the cells to a subsequent exposure to pepstatin A or the chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Pepstatin A-induced desensitization of granule enzyme release and O2- generation appears to be stimulus-specific in that phorbol myristate acetate (PMA) was capable of eliciting normal responses from pepstatin A-pretreated cells. The morphological changes observed in pepstatin A-treated neutrophils are reminiscent of those seen in cells exposed to FMLP.
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PMID:Biochemical, metabolic and morphological characteristics of human neutrophil activation with pepstatin A. 630 51

1-O-Hexadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (C16-AGEPC) and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (C18-AGEPC) stimulated a time- and concentration-dependent release of granule-associated lysozyme and beta-glucuronidase from human neutrophils. Maximum discharge of granule enzymes occurred between 30 and 60 sec after neutrophil exposure to C16- or C18-AGEPC (0.01-10 microM). Less than 10% of total enzyme activity is released when cells are not preincubated with cytochalasin B prior to interaction with the AGEPC analogs. A time-dependent desensitization for granule exocytosis was observed in neutrophils which were stimulated with C18-AGEPC prior to contact with cytochalasin B. The rate and amount of enzyme released by C16- and C18-AGEPC activated neutrophils was significantly enhanced in the presence of extracellular calcium. Trifluoperazine, an inhibitor of calmodulin, caused a dose-related suppression of C18-AGEPC-induced degranulation. Granule enzyme extrusion from C18-AGEPC-treated neutrophils was inhibited by the sulfhydryl reagents, N-ethylmaleimide and iodoacetic acid, and by the glycolytic inhibitor, 2-deoxy-D-glucose. Sodium cyanide was inactive. Pretreatment of neutrophils with C16- or C18-AGEPC rendered the cells unresponsive to subsequent exposure to either AGEPC analog. C18-AGEPC-induced desensitization of neutrophil degranulation appears to be stimulus specific in that serum-treated zymosan and N-formyl-methionyl-leucyl-phenylalanine were capable of eliciting granule enzyme release from C18-AGEPC-pretreated cells.
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PMID:Characteristics of 1-O-hexadecyl- and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine-stimulated granule enzyme release from human neutrophils. 687 58

In vitro, renaturation of reduced and unfolded lysozyme is catalyzed by a mixture of reduced and oxidized glutathione. After initiation of disulfide bond formation associated with the folding process of reduced human lysozyme, molecules have been trapped in a stable form with iodoacetic acid (preserving disulfide bonds) at various times of reoxidation. Each population of molecules trapped in this way was then analyzed by acrylamide gel electrophoresis which separates intermediates on the basis of the number of disulfide bonds they contain and the mean volume of the polypeptide chain. Moreover, the rate of reoxidation of the regeneration mixture was monitored by changes in enzymatic activity, fluorescence quantum yield, and global sulfhydryl group titer. Enzymatic activity was observed to appear after an induction period, and no intermediate, except the fully regenerated species, is active. The first two disulfide bonds reoxidize rapidly, and very few intermediates containing one or two disulfide bonds could be trapped. On the other hand, the intermediates containing three and four disulfide bonds are more predominant, and their formation proceeds more slowly. A folding pathway is suggested, based on the kinetic studies of appearance and disappearance of the various observed intermediates. When these results are compared with those obtained for hen egg white lysozyme and with those found in literature, it can be concluded that the reduced human protein recovers its native conformation more progressively and with more difficulty than the hen egg white protein. This difference might be explained by a greater organization and a greater hydrophobicity in the human molecule.
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PMID:Comparison between the folding of reduced hen egg white lysozyme and that of reduced human milk lysozyme. 715 May 71

A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23 degrees for 40 min. The products are reacted with [1-14C]iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-14C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-14C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.
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PMID:Preparation of a two-disulfide bonded enzymically active derivative from hen egg lysozyme. 744 57

Bacteriolytic enzyme R1 was purified to electrophoretic homogeneity with the recovery of 6.89% activity by ammonium sulfate precipitation, CM-Sephadex C - 50, CM-Sepharose Fast Flow and Sephadex G-75 chromatography from the culture supernatant of Streptomyces griseus RX-17. The molecular weight and PI of R1 were 16.8 kD and 9.10. The optimal temperature and pH for R1 against Streptococcus mutans Ingbritt were 70 degrees C and 6.6, respectively. Below 50 degrees C and at range pH 6 - 10, R1 was stable. While treated at 60 degrees C for 1 hour, the residual activity was only about 20.3%. Zn2+, Cu2+, Fe2+, Cd2+ and Pb2+ could completely inactivate the enzyme. Chelating agents, hydroxylamine hydrochloriae, Monoiodoacetic acid inhibited the lytic activity against Streptococcus mutans Ingbritt, whereas Mg2+, 2-Mercaptoethanol and some surfactants could stimulate the activity. The enzyme had a broad bacteriolytic spectrum against many G+, G- bacteria which were resistant to egg-white lysozyme. Especially high activity was shown on Streptococcus mutans, Staphylococcus aureus and Lactobaillus.
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PMID:[Purification and some properties of bacteriolytic enzyme R1 from Streptomyces griseus RX-17]. 1627 98


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