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)

Chymotrypsin-like activity of multicatalytic proteinase (MCP) purified from human erythrocytes was selectively activated 2.5--3.5-fold by sulfated glycolipids such as galactosylceramide sulfate (SM4) and lactosylceramide sulfate (SM3) but not by other glycolipids including galactosylceramide (GalCer), lactosylceramide (LacCer), GD1a, GM1 and GM3. Heparin also selectively activated trypsin-like activity 2.5-fold, while other mucopolysaccharides did not. This proteinase molecule bound specifically and with high affinity to both SM4 and SM3, but not to GalCer, LacCer and GM3. The binding of SM4 and SM3 to the enzyme molecule was also confirmed by thin layer chromatography.
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PMID:Human erythrocyte multicatalytic proteinase: activation and binding to sulfated galacto- and lactosylceramides. 182 64

Trypsin digestion of pertussis toxin (PT) preferentially cleaved the S1 subunit at Arg-218 without detectable degradation of the B oligomer. The fragment produced, termed the tryptic S1 fragment, appears to remain associated with the B oligomer. Chymotrypsin digestion of PT also preferentially cleaved the S1 subunit without detectable degradation of the B oligomer. The chymotryptic S1 fragment possessed a slightly lower apparent molecular weight than the tryptic S1 fragment and was more accessible to the respective protease. Trypsin- and chymotrypsin-treated PT and PT required the presence of dithiothreitol and ATP for optimal enzymatic activity. Trypsin-treated PT showed approximately a 2-4-fold higher level of expression of ADP-ribosyltransferase and NAD-glycohydrolase activities than PT. Chymotrypsin-treated PT also exhibited approximately a 2-fold greater level of ADP-ribosyltransferase activity than PT. The observed increase in activity of protease-treated PT was due primarily to a shorter time for activation in PT mediated ADP-ribosylation of transducin. In addition, trypsin-digested PT possessed the same cytotoxic potential for Chinese hamster ovary cell clustering as PT. One possible role for the generation of a proteolytic fragment of the S1 subunit of PT would be to produce a catalytic fragment with increased efficiency for ADP-ribosylation of G proteins in vivo.
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PMID:Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit. 185 Jul 38

A new cell line (LC-1/sq) of human lung squamous-cell carcinoma was established from a surgically resected specimen of primary lung cancer. Upon continuous propagation in serum-free culture medium, it secreted trypsin inhibitors into the conditioned medium. The major fraction of the trypsin inhibitor (T1-1) was purified to apparent homogeneity by anion-exchange and gel-filtration high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transblotting to Immobilon. T1-1 effectively inhibited trypsin. Chymotrypsin, plasmin and kallikrein were inhibited to a lesser extent, but urokinase-type plasminogen activator, elastase, thrombin and papain were not inhibited. The activity of T1-1 was acid-stable and heat-resistant, and its molecular weight was 115 kDa by SDS-PAGE. It exhibited single NH2-terminal sequence, and its first 20 NH2-terminal amino-acid residues were identical with those of protease nexin-II (PN-II)/amyloid beta-protein precursor (APP). These characteristics of T1-1 suggest that the major trypsin inhibitor secreted by LC-1/sq is indistinguishable from PN-II/APP. LC-1/sq is the first lung squamous carcinoma cell line that secretes functionally active trypsin inhibitor, PN-II/APP, in vitro and is useful for studying its biological significance in malignant tumor.
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PMID:Establishment of a new human cancer cell line secreting protease nexin-II/amyloid beta protein precursor derived from squamous-cell carcinoma of lung. 191 42

Chymotrypsin treatment of the enterotoxin from Clostridium perfringens results in the loss of 36 amino acids from the N-terminus and 3 amino acids from the C-terminus. This processing results in a 3.2 fold increase in activity on Vero cells, which is close to what had been found for trypsin treatment following the loss of 25 amino acids from the N-terminus.
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PMID:Chymotrypsin treatment increases the activity of Clostridium perfringens enterotoxin. 192 89

Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with Mr 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH2-terminal segment after Arg20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg20 is also slowed by cross-linking Cys15 and Cys90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp19, and elastase cleaves the bond after Ala24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19-24 is highly exposed to the solvent and flexible. The results also indicate that the NH2-terminal region is not directly involved in substrate binding but plays a role in catalysis.
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PMID:Recombinant rat guanidinoacetate methyltransferase: structure and function of the NH2-terminal region as deduced by limited proteolysis. 199 Sep 77

The rate and extent of insulin degradation by trypsin and alpha-chymotrypsin were examined in vitro, and the initial sites of cleavage by alpha-chymotrypsin were identified. The apparent Km for both enzymes was approximately the same but the apparent Vmax for alpha-chymotrypsin was 8.6 times greater. At a molar ratio of 172:1 (insulin:enzyme), chymotrypsin caused near-total loss of insulin within 40 min, while very little insulin was degraded by trypsin. Chymotrypsin appeared to cleave initially at the carboxyl side of the B26-Tyr and A19-Tyr residues, and additional cleavage at the B16-Tyr, B25-Phe, and A14-Tyr residue sites also occurred rapidly. Only two to three other susceptible bonds, which are not exposed at the surface of the insulin molecule, remained intact after the quenching of initial cleavage. Four of the amino acids involved in initial cleavage are essential for receptor binding ability, making it difficult to modify insulin chemically to achieve greater stability without losing activity.
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PMID:Degradation of insulin by trypsin and alpha-chymotrypsin. 206 1

We have obtained evidence that the ligand-recognition region of the integrin beta-subunit, platelet glycoprotein IIIa (GPIIIa), is discontinuous. Receptor function can be localized to residues near the N-terminus and to the central region of the polypeptide chain. The epitope recognized by our monoclonal antibody, CS-1, which substantially inhibits fibrin(ogen) binding to ADP- and thrombin-stimulated platelets [Ramsamooj, Doellgast & Hantgan (1990) Thromb. Res. 58, 577-592], is contained within residues 349-422 of GPIIIa. This sequence is adjacent to a proteinase-resistant domain of GPIIIa which is linked by disulphide bond(s) to an N-terminal segment near to the putative Arg-Gly-Asp recognition site [D'Souza, Ginsberg, Burke, Lam & Plow (1988) Science 242, 91-93]. Limited trypsin digestion of purified platelet GPIIIa yielded a mixture of two-chain molecules comprised of an N-terminal fragment disulphide-bonded to one of four fragments, which began at residues 299, 303, 353 or 423. Tryptic cleavage of the 300-422 segment correlated with loss of immunoreactivity with anti-GPIIIa monoclonal antibody, CS-1. Chymotrypsin cleavage of GPIIIa resulted in an N-terminal 19 kDa fragment joined by at least one intrachain cystine residue to a 46 kDa polypeptide beginning at residue 349. Partial reduction with dithiothreitol released the larger chymotryptic fragment with its epitope for CS-1 intact. These results have enabled us to localize the epitope recognized by our inhibitory monoclonal antibody, CS-1, to residues 349-422 of GPIIIa. Our data are consistent with a structure in which both the N-terminal and central regions of GPIIIa, which may be in close proximity in the functional GPIIb-IIIa complex, participate in ligand binding.
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PMID:Evidence that the central region of glycoprotein IIIa participates in integrin receptor function. 206 10

Antibody blocking studies in the mouse suggest that the MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of inflammation in vivo. We recently showed that chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the MEL-14 antigen from the surface of neutrophils. We report here that chymotrypsin, at low doses (0.1 units/1 x 10(6) cells), but not trypsin, elastase, or collagenase, causes an activation-independent rapid loss (greater than 90%) of the MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions chymotrypsin has no effect on the expression of four other neutrophil surface antigens, including the Mac-1 adhesion protein. Chymotrypsin treatment has no effect on neutrophil adhesion to plastic, migration to C5a, regulation of the Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with chymotrypsin blocks greater than 85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60-70%. These results further support the importance of the MEL-14 antigen in neutrophil extravasation in vivo and indicate that chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of inflammation.
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PMID:Low-dose chymotrypsin treatment inhibits neutrophil migration into sites of inflammation in vivo: effects on Mac-1 and MEL-14 adhesion protein expression and function. 206 57

The molecular structure of the T4 phage tail sheath protein, gp18, was studied by limited proteolysis, immunoblotting, and immunoelectron microscopy. Gp18 is extremely resistant to proteolysis in the assembled form of either extended or contracted sheaths, but it is readily cleaved by proteases in the monomeric form, giving rise to stable protease-resistant fragments. Limited proteolysis with trypsin gave rise to a trypsin-resistant fragment, Ala82-Lys316, with a molecular weight of 27K. Chymotrypsin- and thermolysin-resistant fragments were also mapped close to the trypsin-resistant region. The time course of trypsin digestion of the monomeric gp18 as monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting of the gel revealed that the polypeptide chain consisting of 658 amino acid residues is sequentially cleaved at several positions from the C terminus. The N-terminal portion, Thr1-Arg81, was then removed to form the trypsin-resistant fragment. Immunoelectron microscopy revealed that the polyclonal antibodies against the trypsin-resistant fragment bound to the tail sheath. This supported the idea that at least part of the protease-resistant region of gp18 constitutes the protruding part of the sheath protein as previously revealed with three-dimensional image reconstruction from electron micrographs by Amos and Klug [Amos, L. A., & Klug, A. (1975) J. Mol. Biol. 99, 51-73].
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PMID:Structural studies of the contractile tail sheath protein of bacteriophage T4. 2. Structural analyses of the tail sheath protein, Gp18, by limited proteolysis, immunoblotting, and immunoelectron microscopy. 214 80

Lymphokine activated killer (LAK) cells mediate the lysis of a variety of histologically distinct tumor targets. We investigated the nature and diversity of the structures involved in the recognition phenomenon by evaluating the effects of treating effector and target cells with trypsin and chymotrypsin, enzymes that disrupt surface protein molecules. Chymotrypsin and trypsin treatment of B16 target cells, a murine melanoma cell line, significantly abolished killing by LAK cells. Alternatively, neither of these treatments in P815 cells, a murine mastocytoma cell line, affected killing by LAK cells. Moreover, we found a differential effect of both these enzymes on YAC-1 cells, a murine leukemia cell line, with trypsin having a less inhibitory effect on cytolysis than chymotrypsin. The nature of the LAK cell receptor that presumably plays a role in binding target antigen was also investigated. Treatment of LAK cells with chymotrypsin significantly reduced lysis of the B16 and YAC-1 target cell types. However, trypsin treatment of the effectors only inhibited killing of the B16 tumor cell line. Cytotoxicity exerted against YAC-1 remained unaltered upon trypsinization of LAK cells. These cumulative results indicate heterogeneity of both the receptors on the LAK cells and the surface antigen molecules recognized on these targets. The use of YAC-1 as a target provided us with a tool to compare the LAK with the natural killer (NK) systems. The overall effect of proteolytic enzyme treatment in reducing cell lysis was more pronounced in the NK than in the LAK system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Heterogeneity of cell surface structures involved in cytotoxicity mediated by lymphokine activated killer cells. 218 Oct 73


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