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)

Chymotrypsinogen has been successfully renatured in solution, after reduction of its 5 disulfide bonds in 6 M guanidine-HCl. This has been made possible by the study of the renaturation of a model derivative, polyalanyl-chymotrypsinogen. The reduced derivative is shown to refold and reoxodize spontaneously, with a 30-40% yield, into molecules which are monomeric and fully susceptible to activation by trypsin. Chymotrypsinogen can also be renatured but only in the presence of reagents allowing disulfide interchange and of moderate concentrations of guanidine-HCl or urea. These results illustrate how the kinetic trapping of incorrectly folded molecules by wrong S-S bonds and aggregation can be overcome, thus allowing the correct refolding of the protein.
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PMID:The renaturation of reduced polyalanyl-chymotrypsinogen and chymotrypsinogen. 0 Dec 54

From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin-Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion-exchange chromatography on DEAE-Sephadex A-25. Two of the isoinhibitors accounted for about 50% of the isolated inhibitors and were further purified to a homogeneous state. The isoinhibitors had a molecular weight of about 10000 as determined by molecular-sieve chromatography on Sephadex G-75. They were stable towards extremes of pH and temperatures up to 75 degrees C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HCl. The intact inhibitors were destroyed when the peas were cooked at 100 degrees C or when they were toasted at 130 degrees C. The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20% of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9-8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).
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PMID:The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L.). Purification and properties of the inhibitors. 0 Dec 66

The extracts of granules of human polymorphonuclear leukocytes hydrolyzed a variety of proteins including human and bovine hemoglobin, human fibrinogen, human and bovine serum albumin, bovine elastin, and casein. The hydrolysis of all the proteins except fibrinogen and elastin was increased by addition of urea. Various inhibitors of trypsin, kallikrein, plasmin, Clr, Cls, and other proteolytic enzymes had no inhibitory effect. Slight inhibition was observed with polyanethol sulfonate and strong inhibition with normal human serum. Serum of patients with hereditary angioneurotic edema having no functional C1-esterase inhibitor was as effective in inhibiting the proteolysis as normal serum. The inhibitor was localized in 4S fractions of normal serum fractionated on Sephadex G-200. Fractionation of normal serum by ammonium sulfate precipitation, Sephadex G-200 filtration, and CM-Sephadex chromatography did not result in appearance of inhibitory activity in more than one protein peak, suggesting the possibility that only one inhibitor might be responsible. Since all fractions which contained the inhibitor of proteolysis also contained alpha1-antitrypsin, since sera of patients having low alpha1-antitrypsin levels contained less inhibitory activity, and since antibodies against alpha1-antitrypsin reversed the inhibition obtained from normal serum, the inhibition of proteolysis may be attributed to alpha1-antitrypsin.
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PMID:Some properties of proteolysis by polymorphonuclear leukocyte-granule extracts. 0 49

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.
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PMID:Isolation, chemical, and physical properties of alpha-1-antitrypsin. 0 86

An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.
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PMID:Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands. 0 82

The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH.
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PMID:Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. 0 94

The activity of lecithaniase was determined in 24 strains of Bacillus thuringiensis on four growth media. The highest accumulation of lecithinase was found on the Hottinger medium containing 0.5% of glucose and 0.56% of sodium bicarbonate. Lecithinase appears at the logarithmic growth phase, and its activity is maximal after 10 hours of growth (at the beginning of the stationary phase). Biosynthesis and accumulation of lecithinase occur at pH 6.0 to 9.0. Lecithinase was purified by salting out with ammonium sulphate (75% saturation). Lecithinase is a thermolabile protein; it is stable within pH range of 3.0 to 9.0 and is resistant to the action of trypsin and 8M urea.
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PMID:[Production of lecithinase by Bacillus thuringiensis]. 0 44

Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.
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PMID:Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes. 0 9

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.
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PMID:Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid. 0 17

Platelet-aggregating factor (PAF) was removed from bovine plasma by human platelets fixed with 2% formaldehyde. The degree of adsorption was directly related to the platelet concentration and the length of incubation. Fixed washed platelets (FWP) aggregated with bovine plasma could be deaggregated by 1M KCl, Evans blue, and 8M urea but not by beta-galactosidase. Incubation with 1M KCl eluted some but not all of the PAF, as the deaggregated platelets spontaneously aggregated upon removal of the deaggregating conditions. Also, fixed platelets adsorbed PAF even in the presence of 1M salt or after treatment with Evans blue. Platelet aggregation was not affected by thrombin (20 micron/ml) but was abolished by trypsin at concentrations as low as 4 X 10(-1) microgram/ml. The data suggest that deaggregation is not the result of elution of the loosely bound aggregating factor from the platelet surface, but rather the disruption of noncovalent interplatelet bridging between one or more PAF molecules bound to a specific receptor.
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PMID:Platelet-aggregating factor and the aggregation of fixed washed platelets. 1 45


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