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

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 specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.
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PMID:Proteins of the hepatoma tissue culture cell plasma membrane. 0 57

1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.
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PMID:Isolation and characterization of human plasma alpha 1-proteinase inhibitor and a conformational study of its interaction with proteinases. 0 69

It has previously been shown that mammalian RNA-peptidyl complexes are found in close association with tRNA, but can be separated from the bulk of the tRNA by benzoylated diethylaminoethylcellulose chromatography (Kull, F.J., and Soodak, M. (1971), Biochim. Biophys. Acta 246, l; Gadski, R.A., and Kull, F.J. (1973), Biochemistry 12, 1907). These studies also showed that under aminoacylation conditions the complex fractions were able to act as acceptors for certain amino acids and that the formation of porcine thyroid tyrosyl-complex II was particularly high. Because of this high acceptor function, and because of the importance of tyrosine to thyroid metabolism, further studies were conducted comparing some of the properties of porcine thyroid tyrosyl-complex II with those of porcine thyroid tyrosyl-tRNA. Porcine thyroid tyrosyl-tRNA synthetase was purified in excess of 200-fold and characterized. It was found that maximal aminoacylation was achieved at pH 8.1 in the presence of 150 mM KCl. The Km for tyrosine was determined to be 3.0 X 10(-6) M. The purified thyroid tyrosyl-tRNA synthetase was used under aminoacylation conditions to prepare radioactively labeled porcine thyroid tyrosyl-tRNA and tyrosyl-complex II. Comparisons made using reversed-phase column chromatography (RPC-5) showed distinct differences between the two aminoacylated species and revealed, in addition, a number of isoaccepting forms of tyrosine tRNA. Tyrosyl-complex II was also found to differ from tyrosyl-tRNA in that it is more stable to deacylation at pH 7.0 and at pH 4.4 and to degradation by ribonuclease A. In addition, tyrosyl-complex II, unlike tyrosyl-tRNA, is degraded by trypsin. Ribosomal binding studies showed that tyrosyl-complex II did not respond to the codons for tyrosine, UpApU and UpApC, whereas tyrosyl-tRNA responded to both. It is suggested that thyroid tyrosine complex II is representative of a group of related complexes that constitute the complex II fraction and that, although the complexes resemble tRNA in many respects, they have distinctly different characteristics than conventional tRNA.
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PMID:Thyroid Ribonucleic Acid-Iodopeptides. Comparison of Tyrosyl-Complex II and Tyrosyl-tRNA. 0 30

Rat liver contains two groups of tyrosine aminotransferase (TAT) isoenzymes; during electrophoresis in agar gel one of the groups moves to the anode and the other--to the catode. Cortisol is shown to induce only the anode isoenzymes of TAT, which were isolated, purified and thoroughly analyzed. The inducible anode isoenzyme of TAT spearated from other proteins is more sensitive to the effect of proteases (trypsin and chymotrypsin) than the catode isoenzyme. Some kinetic parameters of the purified TAT isoenzymes were studied. Both isoenzymes have pH optimum around 7.5; their apparent Km values for tyrosine are also similar. However, the catode isoenzyme of TAT possesses a higher affinity for alpha-ketoglutarate than does the anode isoenzyme. Unlike the latter, the former isoenzyme may use oxaloacetate as an amino group acceptor. Pyridoxal phosphate is firmly bound to the catode isoenzyme and can be readily spearated from the anode isoenzyme during dyalisis. An increased sensitivity of the inducible isoenzyme to proteases is due not only to the possibility of coenzyme dissociation, but also to some specific properties of the apoenzyme. The results obtained support the assumption that a high sensitivity of the inducible isoenzymes to proteases provides for a removal of excessive amounts of the enzymes from the cells under cessation of hormonal induction, thus maintaining enzymatic homostasis in the cell.
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PMID:[Isolation and properties of cortisol inducible and cortisol non-inducible isoenzymes of rat liver tyrosine aminotransferase]. 1 43

A method of isolating preparations of pancreatic inhibitor of trypsin, bound with soluble polysaccharide carriers, is worked out. It is demonstrated that the reaction of a pancreatic inhibitor and cyanuric chloride-activated dextran proceeds for OH groups of tyrosine residues and for-epsilon-NH2 groups of lysine residues. A method is offered of the protection of amino groups with citraconic anhydride for the complete retaining of the inhibitory activity during attachment to dextran. Thermic denaturation of pancreatic inhibitor preparations at pH 4.7 and 97 degrees C is studied. It is found that the modification by 2-amino-4.6-dichloro-s-triazine stabilizes the protein molecule, while the interaction with the matrix of soluble dextran does not carry any contribution to thermostability of the pancreatic inhibitor.
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PMID:[Soluble high molecular weight derivatives of trypsin pancreatic inhibitor. Isolation and properties of dextran-bound pancreatic inhibitor]. 2 Jan 63

A soluble somatostatin-binding protein was detected in the cytosol fractions of various rat, human and bovine tissues. Maximum binding occurred at pH8.0-8.5 and was Ca(2+)-dependent. The specific binding of somatostatin per 10mug of cytosol protein from 12 rat tissues ranged between 36 and 15%, and 3% for peripheral blood cells. There was also substantial binding in cytosol from human anterior pituitary and liver, and bovine anterior pituitary. The specific binding in rat and human plasma in the presence of EDTA was only 1%. Gel filtration suggested a molecular weight of approx. 80000 for the somatostatin-binding protein from several sources. Exposure of the binding protein to trypsin eliminates somatostatin-binding activity but ribonuclease and deoxyribonuclease have no effect. The binding protein is thermolabile, ethanol-precipitable, and not completely specific for somatostatin. Bound (125)I-labelled [Tyr(1)]somatostatin is not easily displaced by excess of unlabelled somatostatin. The effects of dithiothreitol and mercaptoethanol on the binding of (125)I-labelled [Tyr(1)]somatostatin to the binding protein suggests that binding involves two sequential steps, first loose binding, then disulphide linkage. Since semipurified somatostatin-binding protein causes a dose-related inhibition of the binding of (125)I-labelled [Tyr(1)]somatostatin in radioimmunoassays for somatostatin, estimates of somatostatin content of tissue extracts by radioimmunoassay in some cases may be spuriously high. It is not yet clear whether the binding protein is a true cytosol protein or an easily solubilized membrane protein.
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PMID:Properties of soluble somatostatin-binding protein. 2 54

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.
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PMID:Activation of fibroblast procollagenase by mast cell proteases. 5 9


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