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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

S-Carboxymethylated L-asparaginase was digested with trypsin and the resulting peptides were isolated by using gel filtration, ion exchange column chromatography and paper chromatography. Among the peptides thus isolated, 27 peptides were considered not to overlap and the sum of the amino acids from these 27 peptides is in good agreement with amino acid composition of the enzyme. The amino acid sequences of the peptides were determined by fragmentation with various enzymes and subtractive Edman degradation.
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PMID:Amino acid sequences of the tryptic peptides from carboxymethylated L-asparaginase from Escherichia coli. 38 70

We show that a non-inhibitory monoclonal antibody (MAB) can be selected that provides substantial and sustained protection against proteolytic inactivation of L-asparaginase by trypsin. Of six non-inhibitory, high affinity, monoclonal antibodies to L-asparaginase, one afforded approximately 70% protection. Inactivation of L-asparaginase is associated with a single cleavage adjacent to lysine-29 that results in loss of an N-terminal fragment with a calculated MW of 2,647. The protective MAB prevented this trypsin cleavage. The products of gene fusions of "humanized" fragments of such antibodies and L-asparaginase could have increased clinical utility.
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PMID:Monoclonal antibodies can protect L-asparaginase against inactivation by trypsin. 136 89

The carboxymethylated L-asparaginase from Escherichia coli A-1--3 was fragmented with cyanogen bromide and the resulting peptides were isolated by using gel filtration on Sephadex G-50 and column chromatography on DE-52. The amino acid sequences of the 7 cyanogen bromide peptides thus obtained were established completely or partially by further fragmentation with trypsin, chymotrypsin and pepsin, and the Dansyl Edman method. Based on the above results and the complete sequences of the tryptic peptides from the carboxymethylated L-asparaginase reported in the previous paper, the whole sequence of the enzyme was established. The reported sequence consists of 321 amino acid residues and its calculated molecular weight is 34 080.
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PMID:The primary structure of L-asparaginase from Escherichia coli. 676 94

Incubation of heat-denatured plasma from the urodele, Amphiuma tridactylum (three-toed amphiuma) or from the anurans Rana ridibunda (European green frog) and Rana catesbeiana (American bullfrog) with either glass beads, porcine pancreatic kallikrein or trypsin did not generate bradykinin-like immunoreactivity. However, peptides were generated in kallikrein-treated amphiuma plasma that contracted vascular rings from the bullfrog systemic arch and had a spasmogenic action on the bullfrog urinary bladder. These peptides which were not generated in trypsin-treated plasma, were purified to homogeneity by reverse-phase HPLC and their primary structures established as: Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp1,Val5]angiotensin II) and Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn1,Val5]angiotensin II). Incubation of synthetic [Asn1,Val5]angiotensin II with amphiuma plasma resulted in deamidation to [Asp1,Val5]angiotensin II. The data suggest, therefore that amphiuma plasma contains an L-asparagine amidohydrolase (asparaginase), as previously described for the eel. Although bradykinin-related peptides have been isolated from frog skin, this study provides evidence tha the kallikrein-kinin system may be absent from the blood of amphibia.
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PMID:Kallikrein generates angiotensin II but not bradykinin in the plasma of the urodele, Amphiuma tridactylum. 759 80

We have demonstrated that a trypsin sensitive enzyme such as L-asparaginase can be rendered trypsin resistant by genetically fusing its gene with that of a single-chain antibody derived from a preselected monoclonal antibody capable of providing protection against trypsin. The chimeric L-asparaginase retained 75% of its original activity upon exposure to trypsin, whereas the native unprotected L-asparaginase control was totally inactivated.
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PMID:Engineering resistance to trypsin inactivation into L-asparaginase through the production of a chimeric protein between the enzyme and a protective single-chain antibody. 764 79

E. coli L-asparaginase was modified with N,O-carboxymethyl chitosan in the presence of normal product L-aspartic acid, which protected the active site of the enzyme. The modified enzyme remained high catalytic activity, showed greater stability against trypsin and alpha-chymotrypsin, but lost its activity more rapidly at high temperature (> 45 degrees C) than did the native enzyme. When tested in vivo, the plasma half-life of the modified enzyme (t1/2 = 40 hr) was over 33 times longer than that of the native enzyme (t1/2 = 1.6 hr). The results showed that the modified L-asparaginase may be much more useful than did the native enzyme for clinical treatments of tumors.
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PMID:The chemical modification of E. coli L-asparaginase by N,O-carboxymethyl chitosan. 892 26

Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C. The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.
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PMID:Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin. 1066 99

In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme.
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PMID:Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis. 1100 28

The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.
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PMID:[Modification of L-asparaginase with colominic acid and the new characteristics of the modified enzyme]. 1105 32

The natural silk sericin recovered from Bombyx mori silk waste by the degumming processing in the high-temperature and high-pressure is a macromolecular protein. Amino acid composition and molecular weight range of the sericin protein as a vector for enzyme immobilization were investigated. The silk sericin protein with different molecular mass from 50 to 200 kDa was poorly soluble microparticles with an average size of about 10 microm. Anti-leukemic enzyme L-asparaginase (L-ASNase) was covalently conjugated on the microparticles of the sericin protein. The immobilized L-ASNase on the natural support by cross-linking with glutaraldehyde maintained 62.5% of the original activity of the enzyme. The Km of sericin-conjugates was 8 times lower than that of native L-ASNase. The bioconjugation of L-ASNase widened the optimum reactive temperature range of the enzyme. The immobilized L-ASNase showed significantly higher stability when the temperature raised to 40-50 degrees C, it also showed preferable resistance to trypsin digestion as compared with native enzyme. The results are discussed regarding the possible explanations of sericin-induced enzyme stability, as well as the possible applications of immobilized L-ASNase research.
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PMID:Immobilization of L-asparaginase on the microparticles of the natural silk sericin protein and its characters. 1502 Jan 51


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