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)

The properties of the functional groups in a protein can be used as built-in-probes of the structure of the protein. We have developed a general procedure whereby the ionization constant and chemical reactivity of solitary functional groups in proteins may be determined. The method may be applied to the side chain of histidine, tyrosine, lysine, and cysteine, as well as to the amino terminus of the protein. The method, which is an extension of the competitive labeling technique using [3H]- and [14C]1-fluoro-2,4-dinitrobenzene (N2ph-F) in a double-labeling procedure, is rapid and sensitive. Advantage is taken of the fact that after acid hydrolysis of a dinitrophenylated protein, a derivative is obtained which must be derived from a unique position in the protein. The method has been applied to the solitary histidine residue of lysozyme, alpha-lytic protease, and Streptomyces griseus (S.G.) trypsin, as well as to the amino terminus of the latter protein. The following parameters were obtained for reaction with N2ph-F at 20 degrees C in 0.1 N KCl: the histidine of hen egg-white lysozyme, pKa of 6.4 and second-order velocity constant of 0.188 M-1 min-1; the histidine of alpha-lytic protease, pKa of 6.5 and second-order velocity constant of 0.0235 M-1 min-1; the histidine of S.G. trypsin, pKa of 6.5 and second-order velocity constant of 0.0328 M-1 min-1; the valyl amino terminus of S.G. trypsin, pKa of 8.1 and second-order velocity constant of 0.403 M-1 min-1. In addition, the results obtained provide clues as to the microenvironments of these functional groups, and indicate that the proteins studied undergo pH-dependent conformational changes which affect the microenvironment, and hence the chemical reactivity of these groups.
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PMID:A competitive labeling method for the determination of the chemical properties of solitary functional groups in proteins. 0 42

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

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins alpha2-macroglobulin and a smaller component of mol.wt. approx. 40000; alpha1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.
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PMID:A neutral collagenase from human gastric mucosa. 0 57

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

Several mucolytic agents were evaluated on sputum for testing their viscolytic activity and the bacterial tollerance to each of them. Proteolytic enzymes (trypsin, pepsin, papain, pancreatin), KJ, and dithiothreitol (or its derivatives) were better tollerated by common respiratory pathogens (H. influenzae, D. pneumoniae, Klebsiella, etc.) than other mucolytic agents, as acetil-cysteine, cisteamine-HCl, tension active substances, mercaptoethanol, and others. The dithiothreitol showed also one of the strongest viscolytic effect and therefore it was selected for the routinary sputum digestion at the concentration 0.1% in PBS pH 7.2. Such a solution was added to sputum specimen in different proportions according to the macroscopic "apparent" viscosity of each specimen. However researches on the comparative viscolytic activity of all the agents hereinafter considered are still in progress.
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PMID:[Study on the viscolytic activity of the sputum (author's transl)]. 1 42

An immunopeptide bearing a3 allotypic determinant(s) was isolated from the gamma chain of an a3 homozygous rabbit (G222-2) immunized with type III pneumococcal vaccine. Immunocogical properties of peptides were studied using a radioimmunoassay that involved inhibition by these peptides of a reaction between 125I-labeled anti-a3 antibody and Sepharose-bound a3 immunoglobulin G (IgG). The gamma chain was isolated from IgG of restricted heterogeneity and then citraconylated and digested with trypsin. The tryptic digest (TD1) was passed through an anti-a3 immunoabsorbent column either directly or after an intermediate step of Sephadex G-75 chromatography. The bound peptides (T1) were eluted with 0.1 M acetic acid and further digested with trypsin. The digest (TD2) was again run on the anti-a3 immunoabsorbent column to purify the bound immunopeptide T2. In the radioimmunossay this immunopeptide was found to have major a3 determinant(s). Its molecular weight was found to be approximately 6,000, which decreased to about 3,000 after reduction and alkylation. These data, together with NH2- and COOH-terminal analyses and cysteine peptide mapping, demonstrated that T2 is composed of two polypeptide chains linked by a disulfide bond, one from the cysteine 22 region having lysine at the COOH terminus and the other from the cysteine 92 region arginine at the COOH terminus. The lysine peptide was separated from the arginine peptide and its NH2-terminal sequence was found to be Gly-Asx-Glx-Ser-Thr-Cys. Since the cysteine is at position 22, the lysine peptide starts at position 17. It has approximately 22 residues. The framework sequence from 17 to 20 is different from those reported so far. In addition, the heavy chain used in these studies has some other unusual features including a histidine, probably in the first hypervariable region. The presence of histidine in the first hypervariable region of rabbit heavy chain has not been reported previously. The other peptide which is about 30 amino acids in length and ends with arginine 94, probably includes positions 67, 70, 71, 84, and 85 that are believed to have substitutions correlating with a allotypes. In a hypothetical three-deminsional model of the Fv portion of rabbit anti-SIII antibody BS-5, residues 17 to 33 of the lysine peptide and 67 to 79 and 84 to 85 which may be present in the arginine peptide are fully exposed on the surface and are far removed from the antibody combining site.
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PMID:Studies on the structural localization of rabbit H chain allotypic determinants controlled by the a locus. Purification and immunological properties of an immunopeptide bearing a3 allotypic determinants. 6 65

Protease-like activity which split plasminogen-free fibrin was demonstrated in 2 M KSCN extracts of the lung and spleen of conventional rats. The activity was virtually undetectable in tissue extracts from germ-free rats. The extracts from the conventional rat tissues split fibrin and fibrinogen remarkably at neutral pH, but not casein, when examined using fibrin, fibrinogen-agar and casein-agar plates. The fibrinolytic activity was inhibited by STI and DFP, indicating a serine protease nature. The activity was not inhibited by TLCK, t-AMCHA or dansyl-L-arginine-methylpiperidine amide (a selective synthetic thrombin inhibitor, OM189). It was neither activated nor inhibited by cysteine, KCN or iodoacetic acid. The results obtained indicate that the protease-like activity of the lung and spleen extracted with 2 M KSCN from conventional rats has properties which differ from those of trypsin, plasmin, plasminogen-activator, thrombin, and cathepsin A, B and C.
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PMID:Fibrinolytic activity of lung and spleen extracts observed in conventional but not in germ-free rats. 9 68

A high molecular weight glycoprotein consisting of three disulfide-linked 142,000 molecular weight chains has been isolated from human blood platelets. The glycoprotein, designated thrombospondin, is released by platelets in response to thrombin treatment and is proteolyzed when left in the presence of platelets after liberation. It is relatively insensitive to degradation by thrombin. Thrombospondin is a filamentous protein of dimensions approximately 7 X 70 nm and contains 1.9% neutral sugars, 1.4% amino sugars, 0.7% sialic acid, and no hexuronic acid. Amino acid analysis reveals that the level of cysteine is approximately 260 residues per molecule. Thrombospondin binds to immobilized heparin but is released by 0.45 M sodium chloride. A single band is obtained by isoelectric focusing, indicating a pI of 4.7 as well as a relatively high degree of purity. Degradation of the intact molecule with trypsin yields a stable core particle of molecular weight 210,000 comprised of three 70,000 chains.
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PMID:Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. 10 49

The reduced and either aminothylated or carboxymethylated H-chain of the monoclonal IgA1 immunoglobulin Tro was digested with trypsin. The tryptic peptides were isolated by gel and ion-exchange chromatography. Because of different methods of alkylation, the cysteine-containing peptides could be obtained in two forms and showed additional overlaps. Sequence studies performed with these fragments elucidated the primary structure of the protein.
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PMID:[Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), III. Isolation and characterization of the tryptic peptides of the H-chain (author's transl)]. 10 14

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.
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PMID:Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. 12 60


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