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 lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.
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PMID:Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101. 178 1

In the synthesis of new prodrugs with inhibitoring action of tumour growth, a new nitrogen mustard derivative was obtained, proceeding of the coupling between an egg-white lysozyme with an antitumor amine nucleophile, the methyl ester of p-bis-(2-chloroethyl)amino-L-phenylalanine (Melphalan), catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), at pH 5.0 and room temperature. In that case, the mechanism for the modification isn't selective of Asp101 in lysozyme. As in cases of histamine and D-glucosamine [3], it is evident that Melphalan is one type of amine who doesn't cause a selective modification of Asp101 but causes somewhat random reaction, because Asp101 is modified followed by modifications of other carboxyls. In this case, we suggest that the amine (Melphalan) may also bind to the substrate binding site in competition with EDC. With this type of amine, enzyme-nucleophile interactions predominate, and the selective activation of Asp101 by EDC is reduced to lead a more random reaction.
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PMID:[Reaction of carbodiimide for the introduction of p-bis(2-chloroethyl)amino-L-phenylalanine to lysozyme]. 279 24

In the cross-linking reaction of lysozyme between Leu129 (alpha-COO-) and Lys13 (epsilon-NH3+) using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from alpha to beta between Asp101 and Gly102 was greatly reduced by addition of beta-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)3]. When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the alpha-carboxyl group of Leu129 was modified with the amine was newly obtained. On the other hand, when ammonia was added, the beta-carboxyl group of Asp119 instead of the alpha-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp119 and Arg125 besides that between Lys13 and Leu129, is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the alpha-carboxyl group reduced the activity to approximately 90% of that of native lysozyme. Des-Leu129 lysozyme, which lacks Leu129, also showed approximately 90% of the activity of native lysozyme. Therefore, the salt bridge between Lys13 and Leu129 may play some role in maintaining the active conformation of lysozyme.
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PMID:Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of Leu129 or Asp119. 350 4

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.
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PMID:Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction. 371 Oct 72

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101. 409 46

The salt bridge between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme was converted to an amide bond by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) reaction in the presence of imidazole (0.3-1 M) at pH 5 and room temperature, followed by dialysis at pH 10. Absence of imidazole under a similar condition did not give this intramolecularly cross-linked lysozyme derivative (CL-lysozyme) but resulted in the formation of intermolecularly cross-linked lysozyme oligomers. From the mechanistic studies on the formation of CL-lysozyme, imidazole was suggested to play the following three roles. (1) Some carboxyl groups activated by EDC in lysozyme were converted to acylimidazole groups which protected them from the reaction with amino groups in other lysozyme molecules at pH 5. These could be hydrolyzed at pH 10 to regenerate free carboxyls. (2) High concentrations of imidazole (pH 5) increased the ionic strength of the solution which weakened the salt bridge in lysozyme and facilitated the activation of the alpha-carboxyl group by EDC. (3) The alpha-carboxyl group activated by EDC was converted to an acylimidazole group which could react with the epsilon-amino group of Lys-13 in the same molecule to form an amide bond. The last step may involve some conformational change of the backbone of lysozyme and be slower than the hydrolysis reaction of the alpha-carboxyl group activated by EDC itself. However, acylimidazole groups are stable against hydrolysis at pH 5. This may afford enough time to allow the epsilon-amino group of Lys-13 to attack the acylimidazole group of Leu-129.
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PMID:Intramolecular cross-linkage of lysozyme. Imidazole catalysis of the formation of the cross-link between lysine-13 (epsilon-amino) and leucine-129 (alpha-carboxyl) by carbodiimide reaction. 641 13

A general procedure which selectively introduced a nucleophilic group at a particular location in the active site of lysozyme has been developed. The coupling of hen egg white lysozyme with amine nucleophiles by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was studied at pH 5 and room temperature. In the presence of an amine nucleophile, such as ethanolamine, ethylenediamine, methylamine, or 4(5)-(aminomethyl)imidazole, the carboxyl side chain of aspartic acid-101 in lysozyme was selectively modified by using a small excess of EDC. The reactivity of Asp-101 is probably due to the specific binding of EDC to the substrate binding site close to Asp-101. With histamine or D-glucosamine, the selectively of Asp-101 was somewhat decreased. This may be due to the specific binding of these amines to lysozyme in competition with EDC, causing a decrease of the selective activation of Asp-101 by EDC. Depending on the amine employed, the lysozyme derivatives obtained exhibited decreased activity (83-52% of native enzyme), suggesting that the modification of Asp-101 weakened substrate binding.
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PMID:Selective modification of aspartic acid-101 in lysozyme by carbodiimide reaction. 729 53

An improved approach for the surface modification of poly(dimethylsiloxane) (PDMS) using carboxymethyl cellulose (CMC), carboxymethyl beta-1,3-dextran (CMD), and alginic acid (AA) was investigated. The PDMS substrates were first oxidized in a H(2)SO(4)/H(2)O(2) solution to transform the Si-CH(3) groups on their surfaces into Si-OH groups. Then methacrylate groups were grafted onto the substrates through a silanization reaction using 3-(trimethoxysilyl)propyl methacrylate. Sequentially, cysteamine was conjugated onto the silanized surfaces by the reaction between the thiol and methacrylate groups under 254 nm UV exposure. Afterward, the amino-terminated PDMS substrates were sequentially reacted with CMC, CMD, and AA in the presence of N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, resulting in the grafting of polysaccharides onto PDMS surfaces. The composition and chemical state of the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS). In addition, the stability and dynamic characteristics of the polysaccharide-grafted PDMS substrates were investigated by XPS and temporal contact angle experiments. A protein adsorption assay using bovine serum albumin (BSA), chicken egg albumin, lysozyme, and RNase-A showed that the introduction of CMD and AA can reduce the adsorption of negatively charged BSA and chicken egg albumin, but increase the adsorption of the positively charged lysozyme and RNase-A. However, CMC-modified PDMS surfaces showed protein-repelling properties, regardless of whether the protein was positively or negatively charged. A cell culture and migration study of glioma C6, MKN-45, MCF-7, and HepG-2 cells revealed that the polysaccharide-modified PDMS greatly improved the cytocompatibility of native PDMS.
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PMID:Photocatalyzed surface modification of poly(dimethylsiloxane) with polysaccharides and assay of their protein adsorption and cytocompatibility. 2061 27

An in situ chemical surface modification method to attach heparin to the inner lumen of a single hollow fiber poly(ether sulfone) (PES) membrane incorporated into a commercial microdialysis sampling device is described. The immobilization process uses gentle, room-temperature conditions with the enzyme laccase (E.C. 1.10.3.2) and 4-hydroxybenzoic acid (4HBA). The resulting functionalized inner membrane surface with a carboxylic acid functional groups allowed for (1-ethyl-3-(3-(dimethylamino)propyl) carbodimide)/ N-hydroxysuccinimide) EDC/NHS chemistry to attach heparin to the membrane surface. X-ray photoelectron spectroscopy measurements suggested successful attachment of 4HBA polymers and heparin onto the PES membrane. The microdialysis extraction efficiency after membrane surface modification was measured with model compounds fluorescein isothiocyanate (FITC)-labeled dextrans and lysozyme and the cytokines acidic fibroblast growth factor (aFGF) and CXCL1 (KC/GRO). This work demonstrates an in situ method to modify commercially available PES hollow fiber microdialysis membranes with amine or carboxylic acid functional groups.
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PMID:In Situ Inner Lumen Attachment of Heparin to Poly(ether sulfone) Hollow Fiber Membranes Used for Microdialysis Sampling. 2958 90