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)

Membrane-bound Na, K-ATPase was digested with trypsin in the presence of Rb+ to form the stable 19-kDa and smaller fragments of the alpha-chain known to preserve occlusion of Rb+ (K+) or Na+. The trypsinized membranes obtained from pig kidney and shark rectal gland were analyzed by electron microscopy. Tryptic digestion preserved general membrane structure but removed both the surface particles observed by negative staining and the protruding cytoplasmic portion of the alpha-subunit identified in thin sections. However, intramembrane particles defined by freeze-fracture were preserved after trypsinization suggesting that the remaining membrane spanning protein fragments retain the native structure within the lipid bilayer after proteolysis.
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PMID:Ultrastructure of membrane-bound Na, K-ATPase after extensive tryptic digestion. 839 37

Thrombin displays remarkable specificity, effecting the removal of fibrinopeptides A and B of fibrinogen through the selective cleavage of two Arg-Gly bonds between the 181 Arg/Lys-Xaa bonds in fibrinogen. Significant advances have been made in recent years towards understanding the origin of the specificity of cleavage of the Arg16-Gly17 bond of the A alpha-chain of human fibrinogen. We have previously proposed a model for the bound structure of fibrinopeptide A7-16 (FPA), based upon NMR data, computer-assisted molecular modeling and the synthesis and study of peptidomimetic substrates and inhibitors of thrombin. We now report the structure of the ternary complex of an FPA mimetic (FPAM), hirugen and thrombin at 2.5 A resolution (R-factor = 0.138) and specificity data for the inhibition of thrombin and related trypsin-like proteinases by FPAM. The crystallographic structures of FPA and its chloromethyl ketone derivative bound to thrombin were determined. Although there are differences between these structures in the above modeled FPA structure and that of the crystal structure of FPAM bound to thrombin, the phi, psi angles in the critical region of P1-P2-P3 in all of the structures are similar to those of bovine pancreatic trypsin inhibitor (BPTI) in the BPTI-trypsin complex and D-Phe-Pro-Arg (PPACK) in the PPACK-thrombin structure. A comparison between these and an NMR-derived structure is carried out and discussed.
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PMID:The structure of a designed peptidomimetic inhibitor complex of alpha-thrombin. 841 74

The heterodimeric VLA-4 integrin has been implicated in lymphocyte migration to inflamed peripheral tissues, lympho-haemopoiesis and autoimmune disease. To determine the structure and function of VLA-4 in physiological processes, molecular forms of the VLA-4 alpha-chain were analysed during T-cell activation. The results showed that prolonged activation of human peripheral T cells was associated with increased cleavage of the 150,000 MW alpha 4 chain into 80,000 and 66,000 MW fragments. Similar-sized alpha 4 fragments could also be generated from 150,000 MW alpha 4 on intact resting T cells by brief trypsinization, whereas trypsin treatment of isolated 150,000 MW alpha 4 resulted in nearly complete protein degradation. Native 80,000 and 66,000 MW alpha 4 chains on activated T cells could not be digested further by trypsin. These results indicated that specific cleavage of 150,000 MW alpha 4 was largely dependent on the tertiary structure of native alpha 4 chains. To determine the specific cleavage site in alpha 4 on peripheral T cells, VLA-4 was isolated and purified from in vitro-activated T cells and the N-terminus of the 66,000 MW fragment was partially sequenced. The sequence SKR/STE was identified as the specific alpha 4 cleavage site on T cells. These results indicate that T cells, upon activation, acquire an enhanced dipeptidase processing activity, which cleaves alpha 4 at a specific site.
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PMID:Specific cleavage of the alpha 4 integrin associated with activation of peripheral T lymphocytes. 843 5

Spectrin Oran (alpha II/21) has been reported previously as a variant of the alpha II domain. Its expression level is low (10% of total spectrin) in heterozygotes denoting a major disadvantage of the mutated alpha-chain dimer or tetramer with respect to their normal counterparts. Spectrin Oran is associated with symptomatic elliptocytosis in the homozygous state. A 1-minute digestion time allowed to perceive a fast trypsin cleavage (not existing normally) after Arg 890 (helix 3 of repeating segment alpha 9). The responsible change was the lack of amino acids 822 to 862 (helix 2 of repeating segment alpha 8). Such a situation fits with the phasing of spectrin according to which mutated helix 2 and distorted helix 3 are adjacent to one another. The internal position of the structural change accounts for the slight self-association defect. The ultimate genetic lesion was a G to A substitution (intronic position-1) in the acceptor splice site of intron 17 resulting in skipping of exon 18. The substitution also created an acceptor splice site 1 base downstream, but the latter was used at a low grade.
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PMID:A splice site mutation of alpha-spectrin gene causing skipping of exon 18 in hereditary elliptocytosis. 849 Jan 86

A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.
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PMID:Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds. 872 12

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.
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PMID:Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases. 884 68

When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal alpha-chain of H+,K(+)-ATPase occurred without any detectable phosphorylation in other regions of the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone trypsin treatment followed by reverse-phase column chromatography yielded three radioactive peptide peaks. The first peak contained both Tyr10(32P) and Tyr7(32P) and the second peak contained Tyr10(32P). The third peak contained Ser27(32P) which was also obtained after trypsin treatment of partially purified H+,K(+)-ATPase preparations phosphorylated with protein kinase-C + Ca2+ or protein kinase-A. This is the first demonstration of Ca2(+)-dependent phosphorylation of the alpha-chain of H+,K(+)-ATPase by protein kinases.
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PMID:Ser-27, Tyr-10 and Tyr-7 in the alpha-chain of pig stomach H+,K(+)-ATPase as Ca(2+)-dependent phosphorylatable sites by intrinsic and extrinsic protein kinases. 888 14

The G2 (A2B1a) glycinin subunit from soybean (Glycine max L. Merr.) was purified and renatured to the homohexameric holoprotein. This protein along with purified beta-conglycinin were subjected to limited proteolysis by trypsin. The generated polypeptide fragments were separated via SDS/PAGE and the amino acid sequence of the N-terminals was determined. Four cleavage points were detected in the alpha-chain A2 of glycinin as well as in the alpha'-chain of beta-conglycinin. From the known three-dimensional structure of 7S globulin and the hypothetical model of 7S globulin-like 11S globulin structure, it was possible to draw the conclusion that two distinct types of susceptible sites for proteolytic cleavage are characteristic of the subunits of both globulins. The first includes the sequences linking N- and C-terminal domains of both globulins and the sequence of N-terminal extensions of 70-kDa subunits from the vicilin-like 7S globulins. The second type includes the loop between beta-strands E and F of the N-terminal domain of 11S globulins and of the C-terminal domain of 7S globulins. A statistically significant similarity was found between the N-terminal extension of the alpha'-chain of beta-conglycinin and the interdomain linker regions of soybean glycinin and pea legumin. It is proposed that the three sequence regions which form the first type of susceptible sites are of similar structural function and might have evolved from the N-terminal segment of a putative single-domain ancestor.
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PMID:Limited proteolysis of beta-conglycinin and glycinin, the 7S and 11S storage globulins from soybean [Glycine max (L.) Merr.]. Structural and evolutionary implications. 889 10

We previously reported finding a novel trypsin-like enzyme in sputum samples from patients with chronic airway diseases, and named it human airway tryptase (HAT). We also obtained data suggesting that HAT is secreted from submucosal serous glands onto the mucous membrane of the airway, and that fibrinogen is cleaved by HAT. We studied whether HAT can act as an anticoagulant in the airway by breaking down fibrinogen. The concentration of fibrinogen in sputum samples was measured by enzyme-linked immunosorbent assay. The concentration was higher in mucoid sputum from patients with bronchial asthma (6.3 +/- 5.5) than in mucoid sputum from patients with chronic bronchitis (1.9 +/- 1.1), and it was higher in purulent sputum from patients with bronchiectasis (18.8 +/- 8.8) than in mucoid sputum from patients with asthma. The trypsin-like activity in sputum (milliunit/ml) was 23.4 +/- 18.0 in mucoid sputum from patients with chronic bronchitis, 46.9 +/- 43.9 in mucoid sputum from patients with asthma, and 14.9 +/- 8.23 in purulent sputum from patients with bronchiectasis. The effect of HAT on human fibrinogen at concentrations from 4 to 2000 micrograms/ml was examined at pH 7.4 and 8.6, with purified HAT at concentrations from 0.5 to 10 milliunit/ml. SDS-polyacrylamide gel electrophoresis showed that HAT can cleave fibrinogen. especially the alpha-chain, at low concentrations (4 to 16 micrograms/ml) and at a high concentration (2000 micrograms/ml) of fibrinogen. Exposure of fibrinogen to HAT resulted in the loss of thrombin-induced clotting capacity; the strength of that effect depended on the duration of exposure to HAT and on the concentration of HAT. From these results we postulate that HAT acts at an anticoagulant in the airways, especially on the mucous membrane, by cleaving fibrinogen transported from blood.
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PMID:[Fibrinogenolytic activity of a novel trypsin-like enzyme found in human airways]. 907 Nov 56

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep alpha1-proteinase inhibitor (alpha1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1. 1x10(3) M-1.s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2x10(6) M-1.s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between alpha1PI and alpha2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess alpha1PI and alpha2-macroglobulin, the alpha-chain being cleaved C-terminally and the beta-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.
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PMID:Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts. 916 5


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