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)

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.
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PMID:Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes. 6 76

The biochemical and functional characterization, and the regulation of plasma membrane expression of the leucocyte tyrosine phosphatase CD45, have been investigated in neutrophils from healthy donors and patients undergoing haemodialysis. CD45 proteins of 180 kD and 130-150 kD were precipitated from neutrophils from both healthy subjects and haemodialysed patients. Prolonged storing, as well as trypsin treatment of samples containing the 180-kD CD45 protein, generated the 130-150-kD polypeptides. The 130-150-kD CD45 polypeptides carried extracellular CD45 epitopes, including the sialic acid-related UCHL1 epitope (CD45RO). Furthermore, these trypsin-generated CD45 polypeptides did not possess phosphatase activity, which could be detected on the 180-kD protein. A remarkable quantitative increase of cell surface expression of the neutrophil CD45 components was detected both after in vitro neutrophil activation and after dialysis treatment with neutropenic membranes. The CD45 biochemical pattern did not qualitatively change upon either in vitro or in vivo dialysis-induced neutrophil activation. The upregulated expression of CD45 on neutrophils from dialysed patients correlated with the neutropenic effect induced by the different dialyser membranes. Maximal upregulation of CD45 expression was observed after 15 min of dialysis with neutropenic membranes, and normal expression levels were restored after 1 h. By contrast, increase of CD45 plasma membrane expression induced in vitro by treatment of normal neutrophils with the degranulatory agents fMLP or Ca2+ ionophore was maintained. These results demonstrate that neutrophil cell surface expression of the 180-kD CD45 protein is upregulated during the in vivo haemodialysis process, and suggest that a proteolytic activity could regulate the enzymatic activity of CD45 by degranulation of its cytoplasmic phosphatase domains.
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PMID:Biochemical and functional characterization of the leucocyte tyrosine phosphatase CD45 (CD45RO, 180 kD) from human neutrophils. In vivo upregulation of CD45RO plasma membrane expression on patients undergoing haemodialysis. 137 Sep 31

A continuous cloned cell line (Y479) was established by culturing normal mouse spleen cells in a high concentration of interleukin-2 (IL-2). Y479 cells showed morphological characteristics of large granular lymphocyte with the phenotypes of Thy-1.2+, T3+, Lyt-1-, Lyt-2-, L3T4-, B220-, AsGM1+, LFA-1+, and TcRV beta 8-. The Y479 cells required a high concentration of IL-2 for their growth but did not express detectable p55 IL-2 receptor (IL-2R) although they bound IL-2 with high and low affinities. Analysis of the IL-2 binding proteins on the Y479 cells revealed that both the high and low affinity receptors consisted only of 70 kDa protein. Analysis of the 70 kDa protein was performed using five monoclonal antibodies (L15, L20, L23, L34, and L61) against human recombinant IL-2. Although they recognized different epitopes, all monoclonal antibodies immunoprecipitated 70 kDa IL-2R that was cross-linked with radioiodinated IL-2. The supernatant after immunoprecipitation with L61 still contained IL-2/IL-2R complex that was L23-reactive, and the supernatant after immunoprecipitation with L23 contained L61-reactive IL-2/IL-2R complex, whereas L15 immunoprecipitated almost all the complex. Limited digestion of IL-2-cross-linked Y479 cells with trypsin caused the liberation of 45 kDa IL-2R fragment cross-linked with IL-2. This complex was immunoprecipitated by L15 or L61 but not by L23. These results suggest that there are at least two distinct 70 kDa IL-2R on the surface of Y479 cells.
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PMID:Two distinct P70 interleukin-2 receptors on a murine large granular lymphocyte clone Y479. 179 Oct 35

The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.
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PMID:Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes. 257 66

Antibodies to most cytolytic T lymphocyte (CTL) external membrane antigens have no effect on CTL-mediated killing in the absence of complement. However, antibodies which do inhibit killing have now been identified for 7 distinct molecular sites. Antibodies to 6 of these "lymphocyte function-associated antigens" (LFAs, also called "blocking sites") inhibit when bound to the CTL, and to the 7th, when bound to the target cell. Mouse homologs have been identified for only 4 of the 7 human LFAs. 5 (probably 6) of the blocking sites inhibit by interfering with adhesion formation between the CTL and the target cell; the exception is T3. None of the presently identified blocking sites are believed to be lethal hit structures (CTL "toxin"). Reduction of target cell H-2 alloantigen density by pretreatment with papain reduces CTL-target functional "affinity", and increases susceptibility to inhibition 100-fold for anti-Lyt-2,3 and 10-fold for anti-LFA-1. This is consistent with the hypothesis that Lyt-2,3 aids in recognition of class 1 MHC antigens, perhaps by strengthening intercellular adhesion. On the other hand, LFA-1 appears to function differently. Trypsin pretreatment of target cells has little effect on MHC antigens or CTL-target affinity, yet still increases by 10-fold susceptibility to inhibition by anti-LFA-1. This is seen in both human and mouse CTL systems. These results suggest the existence of a non-MHC target structure which participates in the adhesion-strengthening function of LFA-1, and which is trypsin (and papain) sensitive: the "trypsin-sensitive counter blocker" (TSCB). LFA-3 may be the human TSCB. The roles of these LFAs in intercellular adhesion extend to more general cell adhesions. Anti-LFA-1 and anti-LFA-3 weaken the spontaneous adhesions which form between cells of the human B cell line JY. These homotypic adhesions are not initiated by immunologic recognition. Anti-LFA-1 is more potent at prolonging allograft survival in vivo than are anti-Lyt-2,3, anti-T200, anti-Thy-1, or anti-I-A. Thus, the potent anti-adhesion properties of LFA-1 seen in vitro may lead to useful immunotherapy in the clinic.
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PMID:Lymphocyte function-associated antigens: regulation of lymphocyte adhesions in vitro and immunity in vivo. 389 52

Production of erythrogenic toxin type B by Streptococcus pyogenes strain T19 was found to be strongly dependent on the pH of the cultivation medium. Maximum yields (greater than 100 mg of toxin/1) were obtained at pH 6.0. In contrast no toxin production was serologically detectable at pH values above 6.5. Purified B-toxin was shown to consist of two components when assayed by SDS-electrophoresis. The molecular weight of the two components was estimated to be 30 000 and 12 000. Isoelectric focusing revealed a heterogeneity of the preparation with isoelectric points between 8.0 and 9.0. Streptococcal proteinase precursor was isolated from culture supernatants of strains T19 and B220 by ammonium sulfate crystallization and purification on CM-Sepharose CL 6B. The protein obtained was homogeneous by SDS-gel electrophoresis and had a molecular weight of 44 000. After autocatalytic activation with mercaptoethanol two bands appeared corresponding to molecular weights 30 000 and 12 000. Isoelectric focusing of proteinase precursor preparations yielded a double band at pI 8.2-8.3. However, activation of precursor to active proteinase finally resulted in a change of the pI to 9.0. Erythrogenic toxin type B, streptococcal proteinase precursor, its intermediate activation products and the active proteinase itself reacted serologically identical with anti B-toxin antiserum. Streptococcal proteinase precursor provoked a delayed skin reaction and was pyrogenic as well as mitogenic. Its pyrogenic activity could be inhibited by antiserum against scarlet fever toxin (Wellcome Laboratories). We therefore believe erythrogenic toxin type B to be identical with streptococcal proteinase precursor. This helps to understand the heterogeneity of B toxin, its inactivation by trypsin and the different protocols for toxin production described in the literature.
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PMID:Isolation and characterization of erythrogenic toxins. V. Communication: identity of erythrogenic toxin type B and streptococcal proteinase precursor. 635 75

A monoclonal antibody, 9.1C3, was used to investigate the mechanism of natural killer (NK) cell-mediated lysis. In addition to blocking NK cell function, the antibody blocked antibody-dependent cellular cytotoxicity against the K562 target cell at the effector cell level. The stage at which 9.1C3 antibody inhibited cytolysis was established with a Ca++ pulse technique, whereby it was shown that the antibody inhibited killing at a discrete step after the Ca++-dependent programming for lysis. The 9.1C3 antigen appeared to be associated with the T200 glycoprotein complex. Thus the 66 and 77 Kd proteins detected by 9.1C3 were also precipitated with a monoclonal antibody to T200, and in sequential immunoprecipitations, 9.1C3 antibody removed these bands from immunoprecipitates with antibody to T200. Also, in co-modulation studies, it was found that antibody to T200 co-capped the 9.1C3 antigen but that capping with 9.1C3 antibody did not induce co-modulation of the T200 antigen. Expression of the 9.1C3 and T200 antigens on different cell types, however, was not identical, and the 9.1C3 antibody did not immunoprecipitate high m.w. proteins in the region of 200 Kd. Functionally, in NK cell killing studies, the antibody to T200 used alone did not block but was synergistic with the 9.1C3 antibody. The differential effect of the enzymes pronase and trypsin on the cell surface expression of the 9.1C3 and T200 antigens reflected the ability of these enzymes to inhibit NK cell killing. These data suggest that the 9.1C3 antigen participates in a late event in the cytolytic pathway.
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PMID:A novel antigenic cell surface protein associated with T200 is involved in the post-activation stage of human NK cell-mediated lysis. 637 48

Very low doses of trypsin (5 micrograms/ml) are sufficient to ablate NK cell activity. This finding was used to make several observations, and we have attempted to relate these observations to specific cell surface macromolecules. First, trypsinized effector cells no longer lysed seven different NK-susceptible targets, but the lysis of three additional targets was unaffected. These results suggest a heterogeneity of recognition potential that is inconsistent with the notion that there is only one class of NK "receptors" and one class of "target structures." Trypsin does not affect the conjugation of effector and target cells. Secondly, we have tried to identify those cell surface molecules that are affected by this low dose of enzyme. The examination of the 125I-labeled glycoprotein fraction NK-enriched cells showed that at least four molecules are cleaved, one of which may be in the T200 family. The examination of the [3H]galactose-labeled cell surface glycoproteins suggested in particular that some high m.w. glycoproteins were affected at the dose of trypsin that ablates NK function. Analysis of those molecules that we previously implicated in NK function, defined by monoclonal antibodies that block NK lysis, allowed us to rule out a role for the Tp 50 and Lp95-150 structures, while providing additional evidence of a role for the T200 glycoproteins in the trypsin-sensitive stage of cytolysis. Finally, closer examination of the electrophoretic mobilities and trypsin sensitivity of the T200 structures on highly enriched NK cells showed these structures to be indistinguishable from the T cell form of T200, yet quite distinct from the monocyte form. These results are therefore consistent with the possibility that NK cells are of the T rather than the monocyte lineage, and furthermore support a role for the T200 structure in the post-binding trypsin-sensitive stage of the NK cytolytic process.
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PMID:T cell nature and heterogeneity of recognition structures of human natural killer (NK) cells. 641 67

N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK), an irreversible inhibitor of trypsin-like serine proteases, is a potent, nontoxic inhibitor of cytotoxic T lymphocyte (CTL) activity with half-maximal inhibition of an alloreactive CTL clone occurring at [TLCK] = 30 microM. We have utilized TLCK as an affinity probe for functionally important CTL surface molecules by raising rabbit antibodies specific for the tosyl group and employing them as immunoprecipitating reagents. When 125I-labeled cloned CTL were treated with TLCK, immunoprecipitation with rabbit anti-tosyl antibodies and analysis by polyacrylamide gel electrophoresis revealed a small number of TLCK-binding proteins. Prior alkylation of radiolabeled CTL with iodoacetamide inhibited TLCK binding only slightly, suggesting that TLCK binding did not occur via free sulfhydryl groups. Thymocytes and a second CTL clone both had very similar patterns of TLCK-binding proteins; in contrast the TLCK-binding proteins of B cells differed greatly. Sequential immunoprecipitation experiments identified the predominant CTL TLCK-binding protein as T200. Lymphocyte function-associated antigen-1 also reacted with TLCK but to a lesser extent. The inhibitory role of cell-surface bound TLCK (vs intracellular TLCK) was demonstrated by protection experiments using Concanavalin A, a reversible ligand of the CTL cell surface. These experiments suggest that T200 may be required for cytotoxic activity of CTL.
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PMID:The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes. 641 72

T200 glycoprotein, a major cell surface component of murine hematopoietic cells, is a phosphorylated transmembrane glycoprotein. Two distinct regions of the molecule can be defined by radiolabeling with a variety of metabolic precursors or by lactoperoxidase-catalyzed iodination, in combination with protease treatments, immunoprecipitation techniques, and peptide "mapping" analysis. A relative protease-resistant domain, which is exposed on the cell surface and contains the antigenic site recognized by a monoclonal anti-T200 antibody known to react with the exterior cell surface, contains most if not all of the mannose-containing oligosaccharide units of the glycoprotein and all of the amino acid residues labeled by lactoperoxidase-catalyzed iodination of intact viable cells. This protease-resistant fragment migrates with an apparent molecular weight of approximately 100,000 in sodium dodecyl sulfate-polyacrylamide gels. The remaining portion of the molecule contains a region, extensively digested by trypsin, which is exposed on the cytoplasmic side of the plasma membrane and contains phosphoserine residues which can be labeled with 32PO4 in vivo. A 125I-labeled tryptic peptide derived from this region of the molecule was obtained if membrane preparations from cells disrupted by nitrogen cavitation were labeled by lactoperoxidase-catalyzed iodination.
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PMID:Disposition of T200 glycoprotein in the plasma membrane of a murine lymphoma cell line. 735 47


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