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)

The tripeptide Bz-Arg-Gly-Asp(-OMe)-OH was synthesized by enzymatic method. Bz-Arg-Gly-OEt was synthesized by trypsin in ethanol containing 0.1 M Tris/HCl buffer (pH 8.0), and then H-Asp(-OMe)(2) was incorporated into the Bz-Arg-Gly-OEt using chymopapain in 0.25M CHES/NaOH buffer (pH = 9.0, EDTA 10 mM). The yield of Bz-Arg-Gly-OEt and Bz-Arg-Gly-Asp(-OMe)-OH were 80% and 70% using 1M Bz-Arg-OEt and 0.5M Bz-Arg-Gly-OEt, respectively. For Bz-Arg-Gly-OEt synthesis reaction at high concentrations of the substrates, the buffer content in ethanol was a key factor to determine the optimal reaction condition. In Bz-Arg-Gly-Asp(-OMe)-OH synthesis reaction, the yield was low in organic solvent due to various side products such as Bz-Arg-OH, Bz-Arg-Gly-OH, and Bz-Arg-Gly-Asp(-OMe)-Asp(-OMe)-OH, suggesting that chymopapain has a very broad substrate specificity of the S(1) site. The Bz-Arg-Gly-Asp(-OMe)-OH synthesis rate and its yield were dramatically elevated and the side reactions were reduced using only the CHES/NaOH buffer (pH = 9.0, EDTA 10 mM) as a reaction media. The final product Bz-Arg-Gly-Asp(-OMe)-OH was identified to be formed via C-terminal hydrolysis of Bz-Arg-Gly-Asp(-OMe)(2) after the nucleophile, H-Asp(-OMe)(2), was added.
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PMID:Protease-catalyzed tripeptide (RGD) synthesis. 1068 65

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.
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PMID:High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays. 1570 70

An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.
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PMID:Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp. 1656 14

Assay of fractions obtained from ion exchange chromatography of papaya latex on CM Sephadex-C50, size exclusion chromatography on Sephacryl S-300 and size exclusion HPLC have provided an insight into the relative contributions of the gluten-detoxifying enzymes present. This outcome has been achieved by the use of the above chromatographic techniques, coupled with assays of lysosomal activity, protease activity using benzylarginine ethyl ester (BAEE) as substrate, prolyl endopeptidase (PEP) using glycylprolylnitroanilide and a prolidase assay using acetylprolylglycine. These procedures have shown that the activity in papaya latex is due largely to caricain and to a lesser extent, chymopapain and glutamine cyclotransferase. The presence of caricain and these other enzymes was confirmed by mass spectrometry of trypsin digests of the most active fraction obtained by CM Sephadex-C50 chromatography and size exclusion HPLC. Fractions rich in caricain would be suitable for enzyme therapy in gluten intolerance and appear to have synergistic action with porcine intestinal extracts.
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PMID:Papaya latex enzymes capable of detoxification of gliadin. 1915 82

N,N'-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C-C coupling of 2 N-Boc-L-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY-(INH)(2) lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY-(INH)(2) and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.
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PMID:A dityrosine-based substrate for a protease assay: application for the selective assessment of papain and chymopapain activity. 2244 80


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