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)

We described earlier the facilitated purifications of the trypsin and aminopeptidase components present in Pronase (Vosbeck, K. D., Chow, K. -F., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6034). A partially resolved protein mixture left over after one of the steps in that procedure was passed through a Sephadex G-75 column. By this means, a component with carboxypeptidase activity was separated from associated serine endopeptidases. Further purification of this exopeptidase to apparent homogeneity was acheived by refiltration through the same Sephadex column and by CM-cellulose chromatography. A single protein band was observed after acrylamide gel electrophoresis; analysis by sedimentation equilibrium using the meniscus depletion method gave a molecular weight of 30,300. This enzyme demonstrates activity against Nalpha-benzyloxycarbonylglycyl-L-leucine and hippuryl-D,L-phenyllactate; no activity was found against Nalpha-acetyl-L-tyrosine ethyl ester, Nalpha-benzoyl-D,L-arginine-p-nitroanilide, or L-leuckne-p-nitroanilide. The maximum activity lies between pH values of 7 and 8; the enzyme is stable between pH values of 6 and 10. At room temperature 1,10-phenanthroline inactivates the enzyme completely whereas EDTA has no effect. Of the many cations tested, only Co2+, Ni2+, or Zn2+ restores activity to the 1,10-phenanthroline-treated enzyme; Co2+ provided 3 times the native activity. The metal in the native protein was found to be zinc. These findings are similar to those recorded with bovine pancreatic carboxypeptidase A, and suggest the possibility that the present enzyme may ge genetically related to the mammalian protein, as in previously noted examples of homology of three Pronase endopeptidases to pancreatic serine enzymes.
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PMID:Proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Purification and characterization of the carboxypeptidase. 0 Mar 99

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

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.
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PMID:Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis). 0 91

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 metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.
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PMID:Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue. 0 4

5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.
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PMID:Specific fluorescent derivatives of macromolecules. Reaction of dansyl fluoride with serine proteinases. 0 13

The active site of porcine enteropeptidase (EC 3.4.21.9) was investigated in order to characterize better both catalytic and binding sites. The participation of a serine and a histidine residue in the catalytic process was fully confirmed and the two residues were located on the light chain of the enzyme. The binding site was found to be composed of at least 2 subsites S1 and S2. The subsite S1 (similar to the trypsin-binding site) is responsible for the interactions with the small substrates of trypsin and the lysine side chain of trypsinogen, while subsite S2 (probably a cluster of lysines) is responsible for the interactions with the polyanionic sequence found in all trypsinogens. Binding of substrate by subsite S2 led to an increased efficiency of the catalytic site which can be correlated to the known high specificity of enteropeptidase.
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PMID:On the catalytic and binding sites of porcine enteropeptidase. 1 10

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.
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PMID:The reactive site of eggplant trypsin inhibitor. 1 22

The protease isolated jawasee shrub was found to hydrolyze egg albumin, casein, haemoglobin and gelatin optimally near neutral pH. Fibrin, bovin serum albumin, skin albumin and skin mucoids were hydrolyzed at slightly alkaline pH, while skin globulins were hydrolyzed at slightly acidic pH. The enzyme had no effect of fibrous collagen. The optimum conditions for the hydrolysis of 50 mg of egg albumin were found to be 50 mg of alhagain at pH 6.0 and 45 degrees C for 30 minutes. A Km value of 4.4 X 10(-3) M was obtained from the Lineweaver-Burk plot for the hydrolysis of egg albumin. The enzyme was found to be comparatively thermostable and was most stable at pH 4.7. Ultraviolet irradiation exhibited no appreciable effect on the enzyme activity. The ultraviolet absorption spectrum of alhagain in bi-distilled water resembles those of bromelain and trypsin. The sugar-containing enzyme was found to have a molecular weight of 20,650. The enzymeconsists of 189 amino acid residues per molecule, neutral and acidic amino acids being present in high concentrations. The partial specific volume of alhagain was calculated to be 0.743 ml/g from its amino acid composition. Phenylalnine and arginine formed the amino terminal amino acids of alhagain, while aspartic acid and serine were identified as its carboxy terminal amino acids. Results are discussed with relation to other plant proteases.
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PMID:Studies on the physico-chemical properties of alhagain. 2 Nov 47

A trypsin inhibitor was isolated from beans of Phaseolus vulgaris, cultivar. Kintoki, and the specific activity increased 200 times as high as that of the crude extract. It was homogeneous on several electrophoreses and the molecular weight was about 13,000. The amino acid composition was characterized by high ratios of cystine, aspartic acid, and serine. It inhibited trypsin in a molar ratio of 1 : 1 and alpha-chymotrypsin in a molar ratio of 2 : 1. It, however, inhibited neither pepsin nor pronase. It was relatively stable to heat treatment in the acidic medium, but not in the alkaline medium. Neither pepsin nor pronase destroyed the inhibitory function.
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PMID:The isolation and characterization of a trypsin inhibitor from Kintoki bean (Phaseolus vulgaris). 2 95


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