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)

Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances.
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PMID:Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm. 15 36

Dextran-induced agglutination of Streptococcus mutans cells is independent of cell-bound dextransucrase activity. Toluene extraction or the presence of Hg2+ or Cu2+ markedly decreased or completely abolished cell-bound dextransucrase activity without adversely affecting dextran-induced cell agglutination. Cells treated by heating at 100 C until cell-bound dextransucrase was completely inactivated continued to agglutinate when induced by dextran-induced cell agglutination resulted from cell treatment with trypsin and several other enzymes, as well as from ethylenediaminetetraacetic acid treatment, without a corresponding loss of cell-bound dextransucrase activity. Cells possessed a greater avidity for branched dextrans of low molecular weight than for linear dextrans of the same weight, indicating that size alone does not determine the efficiency of dextran as an inducer of agglutination. Divalent metal ions were required for both sucrose- and dextran-induced agglutination of S. mutans K1-R cells. Although normal cells of strain 6715-49 did not appear to require divalent cations for agglutination, heat- and ethlyenediaminetetraacetic acid-treated cells specifically required Ca2+. The role of Ca2+ in cell agglutination may be either to activate the cell-surface dextran receptor or to form specific intercellular Ca2+ bridges.
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PMID:Relationship between cell-bound dextransucrase and the agglutination of Streptococcus mutans. 80 56

A bile salt mixture and pure sodium taurocholate were each shown to increase the esterolytic activity of trypsin in aqueous solution and in intestinal juice. rho-Toluene-sulphonyl-L-arginine methyl ester (TAME) was used as a substrate, and both a spectrophotometric and a potentiometric assay system were used. The maximal potentiation of the esterolytic activity of trypsin by bile salts was about 1.6 to 2.2 times the activity without bile salts (depending on the assay conditions and whether the trypsin was in aqueous solution or intestinal juice). The proteolytic activity of trypsin was decreased by the addition of bile salts. It seemed likely, therefore, that the potentiating effect of bile salts on trypsin esterolytic activity is primarily on the substrate (TAME) rather than trypsin itself. It was thought that TAME might be taken up into bile salt micelles and thus be more readily hydrolysed by trypsin, but we were unable to substantiate this hypothesis. The precision of the trypsin esterolytic assay was better when bile salts were not added. If however bile salts were to be used routinely in the trypsin assay, it would be useful to ensure that the concentration of calcium, included as activator, is sufficiently low to prevent the formation of a precipitate. This precipitate is probably a complex of calcium and bile salts.
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PMID:The effect of bile salts on the esterolytic assay of trypsin. 91 20

A method of covalent immobilization of trypsin on polyurethanes containing residues of 2.4-toluene or hexamethylene diisocyanates in a side chain is developed. It is shown that the amount of the immobilized enzyme is independent of isocyanate nature, but its proteolytic activity is higher on carriers containing 2.4-toluene diisocyanate residues. This is accounted for by higher mobility of enzyme molecules due to a decrease in the number of chemical bonds between trypsin and the polymer matrix. The immobilized enzyme is stable for a long time and does not lose proteolytic activity being cyclically used for many times.
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PMID:[Immobilization of trypsin on polyurethane carriers]. 165 74

Initial rates of peptide-bond synthesis catalyzed by poly(ethylene glycol)-modified chymotrypsin in benzene were determined using high-performance liquid chromatography. Enzymatic synthesis of N-benzoyl-L-tyrosyl-L-phenylalanine amide from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine amide was found to obey Michaelis-Menten kinetics an to be consistent with a ping-pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N-benzoyl-L-tyrosine ethyl ester and N-alpha-benzoyl-L-lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10-fold smaller than that obtained with poly(ethylene glycol)-modified trypsin in the synthesis of N-alpha-benzoyl-L-lysyl-L-phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme-binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.
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PMID:Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents. 265 20

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.
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PMID:Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex. 288 33

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.
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PMID:Functional properties of proteins immobilized on albumin microspheres. 340 60

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.
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PMID:The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. 442 92

Several esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid and alpha-N-toluene-p-sulphonyl-l-homoarginine by alpha- and beta-trypsin were compared. On the basis of values of the specificity constants (k(cat.)/K(m)), the two enzymes display similar catalytic efficiency towards some substrates. In other cases alpha-trypsin is less efficient than beta-trypsin. It is possible that alpha-trypsin possesses greater molecular flexibility than beta-trypsin.
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PMID:Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine alpha- and beta-trypsin. 447 5

The acetylation of the free amino groups of insulin was studied by reaction of the hormone with N-hydroxysuccinimide acetate at pH6.9 and 8.5. The products formed were separated by chromatography on DEAE-Sephadex and were characterized by isoelectric focusing, by end-group analysis, by the incorporation of [(3)H]acetyl groups in the molecule, and by treatment with trypsin that had been treated with 1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one (;tosylphenylalanyl chloromethyl ketone'). Three monosubstituted products, two disubstituted products and one trisubstituted derivative were prepared. The alpha-amino groups of the terminal residues and the in-amino group of the lysine-B29 were the sites of reaction. Acetylation of any of the free amino groups did not affect the biological activity of insulin. It was demonstrated, however, that substitution at the glycine-A1 amino group by the larger residues, acetoacetyl or thiazolidinecarbonyl, produced a decrease in biological activity. Modification of the lysine-B29 or phenylalanine-B1 amino groups with these larger reagents did not affect the biological activity. Modification of the phenylalanine-B1 amino group by any of the three substituents resulted in a large decrease in the affinity of insulin for anti-insulin antibodies raised in the guinea pig. Modification of the other two amino groups did not affect the reaction with antibody. These observations are correlated with the tertiary structure of insulin.
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PMID:The acetylation of insulin. 511 88


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