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)

During the process of cultivation of Th. vulgaris several proteases are formed. In the present investigation the extensively purified major component was used. The substrate specificity was determined by means of 7 proteins, 7 amino acid esters, 5 fatty acid esters and 15 amino acid 4-nitroanilides. Among the protein substrates tested, urea denaturated hemoglobin was split best, followed by gelatin, casein, field bean protein, serum albumin and gluten. The weakest rate of hydrolysis was observed with elastin. In contrast to this acetyl-(L-ala)3-methylester, that is a substrate for elastase, was split best from all the esters tested. Only 8% of this activity could be found with the chymotrypsin substrates acetyl-L-tyr-ethylester and acetyl-L-phe-ethylester and 1% of the above activity with the trypsin substrates tosyl-L-arg-methylester and benzoyl-L-arg-methylester. The fatty acid esters and the p-nitroanilides were hydrolyzed much more slowly. The pH-optimum of thermitase was found in the weakly alkaline region of pH 7 to 9. There were only small differences between the individual high and low molecular substrates. The temperature optimum was between 60 and 75 degrees C for esters and p-nitroanilides as substrates and at 90 degrees C for casein. It should be mentioned that the enzyme was quickly inactivated at temperatures above 70 degrees C.
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PMID:[Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 3. Substrate specificity and properties of partially purified thermitase]. 3 57

The cleavage specificity of a protease from Thermoactinomyces vulgaris (thermitase) was determined by the insulin B-chain and the cleavability of casein and haemoglobin by this enzyme as compared to other proteases (trypsin, chymotrypsin, proteases from Bac. megaterium and cytophages). The most intense splitting effect on the substrates under investigation (insulin B-chain, casein and haemoglobin) is exerted by thermitase, i. e., the unspecificity of this enzyme is especially marked.
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PMID:[Substrate specificity of a protease from Thermoactinomyces vulgaris]. 46 Mar 96

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.
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PMID:Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type. 331 95

In the study of the covalent immobilization of aminoacylase, thermitase, pepsin, trypsin, chymotrypsin, elastase, subtilisin, penicillinamidohydrolase, carboxypeptidase A, cystathionine-beta-synthase, and anticathepsin D-IgG to copolymers of 2-hydroxyethyl methacrylate and ethylene dimethacrylate (Separon HEMA) containing epoxy groups a marked influence of added salts on the immobilization efficiency was observed. Yields in covalently bound active enzymes were dependent on the concentrations and type of ions added, which can be arranged according to the Hofmeister series. At a distinct concentration, the salting-out ions cause a protein-matrix hydrophobic interaction which is a prerequisite for the covalent bond formation.
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PMID:Influence of salts on the covalent immobilization of proteins to modified copolymers of 2-hydroxyethyl methacrylate with ethylene dimethacrylate. 340 61

Casein, wheat gluten or field-bean protein isolate (vicia faba) was hydrolyzed enzymatically by trypsin or thermitase (proteinase from Thermoactinomyces vulgaris). The degree of splitting of the protein peptide bounds was, resp., 18% or 30% for casein, 8% or 30% for wheat gluten and 16% or 29% for the field-bean protein isolate. The peptide pattern of the protein hydrolysates was measured by gel chromatography. The absorption of the enzymatic protein hydrolysates was compared with the absorption of the equimolar mixtures of free amino acids by perfusion of the distal or proximal part of the small intestine of nonanaesthesized rats. The absorption was measured on the basis of the amino acid disappearance from the perfused part of the small intestine and expressed as absorption of the total amino acids, of each single amino acid and as coefficient of variation of amino acid absorption. There are no differences in the total amino acid absorption of the tryptic casein hydrolysate and the three thermitatic protein hydrolysates compared with their corresponding mixtures of free amino acids. The absorption is decreased in case of larger oligopeptides. Their preceding mucosal hydrolysis is slower than the absorption of the splitting products. This is true for tryptic hydrolysates of wheat gluten and field-bean protein. The absorption patterns of the single amino acids are different for protein hydrolysates compared with their equimolar mixtures of amino acids in case of a same total absorption, too. The coefficient of variation is low (more equal amino acid absorption) in hydrolysates consisting of a homogeneous mixture of low molecular weight peptides. It is more increased in mixtures of free amino acids or in hydrolysates containing longer-chain peptides.
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PMID:[Absorption of enzymatic protein hydrolysates and equimolar amino acid mixtures in the perfused small intestine of the rat]. 672 74

Thermitase has been investigated as a means for obtaining single cells from tissue material, for enzymatic detachment of cultivated cells from the substrate and rarification of cells with subsequent passaging of mouse embryonic fibroblasts. The action of this enzyme was compared with that of "trypsin for cell cultivation". Tissue digestion showed that thermitase at 1/50 of the enzyme concentration needed with trypsin, was 1.5 fold more effective in yielding single cells. In cell cultivation thermitase is able to detach cells from the substrate at the same low concentration of 0.05 mg enzyme/ml and to give sufficient rarification, without the need of adding complexing agents. Rate of attachment, cell form and multiplication in subcultures corresponded to those after application of trypsin. The best results in cell detachment and rarification and the most uniform cell morphology were obtained with thermitase at a concentration of 0.025 mg enzyme/ml under addition of 4 mM of complexing agent. At that, thermitase proved 50 fold more effective than trypsin. Another advantage of thermitase is its better storage quality at 4 degrees C in dissolved form.
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PMID:[Use of thermitase in the cultivation of embryonal mouse fibroblasts]. 675 95

The steric arrangements of the amino acyl residues in the catalytic triads and tetrads of the active site are compared with each other by means of systematic analysis of the conformation of the serine proteases stored in the Brookhaven Protein Data Bank. On this basis a differentiation between the representatives of the (chymo)trypsin family on the one hand and those of the subtilisin family on the other hand is found. The enzyme tonin distinguishes from representatives of the (chymo)trypsin family and should be classified to a new subclass of this family. Thermitase represents a new subclass of the subtilisins. The spatial orientation of the amino acyl residues of the active site of tonin suggests a new mechanism of enzyme catalysis that possibly also occurs in dipeptidyl peptidase IV.
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PMID:Classification of serine proteases derived from steric comparisons of their active sites. 814 70

An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated delta G contribution of the amino group from the BPTI residue Lys15 (9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal). In OMTKY3, different binding loop residues are known to affect differently the binding (delta delta G) to alpha-chymotrypsin and protease B; a good qualitative agreement was found between the calculated delta G(residue) estimates and the experimental delta delta G data (correlation coefficient 0.7). Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures. 825 66

N-Protected dipeptides containing L-3-thia-analogues of phenylalanine, p-nitro-phenylalanine, lysine and leucine respectively were prepared applying an enantioselective enzymatic reaction step. Racemic synthetic intermediates of the type acyl-NH-CH(R(1))-CO-D,L-NH-CH(S-R(2))-COOBzl were selectively deprotected at the C-terminus by enzymatic hydrolysis using thermitase or trypsin.
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PMID:Thia-analogues of amino acids synthesis of peptide derivatives containing 3-thia-analogues of amino acids. 2419 93