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)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.
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PMID:Purification and physicochemical characterization of murine T cell replacing factor (TRF). 387 Nov 9

The choriocapillaris is one example of a capillary bed lined by a fenestrated endothelium that is restrictive to exogenous tracers and endogenous plasma proteins. In this study we have examined the distribution of cell-surface monosaccharides utilizing biotinylated lectin-avidin ferritin cytochemistry. Receptors for wheat germ agglutinin were localized to the plasmalemma and diaphragms of some fenestrae, vesicles, and channels at the luminal endothelial front in amounts greater than seen for the other lectins employed. The absence of labeling following inhibition with N-acetylglucosamine and after tissue digestion with N-acetylhexosaminidase, but not after neuraminidase indicated that this lectin marked N-acetylglucosamine residues and not sialic acid. Wheat germ agglutinin receptors were not affected by pronase E or trypsin digestion, but were partially removed by proteinase K. The latter also removed many fenestral diaphragms. Wheat germ agglutinin receptors were cleaved with endoglycosidase D. The combined results indicate that the wheat germ agglutinin receptor is of the low-mannose type and part of a protein with hydrophobic properties. Receptors for concanavalin A (mannose) and Ricinus communis agglutinin (galactose) were also localized to the plasmalemma and endothelial diaphragms. The examination of sections at different tilt angles revealed that these lectins bound to the endothelium in a non-random distribution, encircling diaphragms of fenestrae and channels. Soybean agglutinin (N-acetylgalactosamine) marked endothelial structures sparsely. Following digestion with pronase E or trypsin, receptor sugars for the latter three lectins were completely removed, indicating their presence on protease susceptible glycoproteins. These findings demonstrate that the endothelium of the choriocapillaris bears carbohydrate moieties that are different than those described for permeable fenestrated endothelia.
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PMID:The cell surface of a restrictive fenestrated endothelium. I. Distribution of lectin-receptor monosaccharides on the choriocapillaris. 394 19

The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2-3H]mannose or L-[5,6-3H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2-3H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2-3H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6-3H]glucosamine and L-[1-14C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3H-labeled N-acetylglucosamine and N-acetylgalactosamine. This high molecular weight glycopeptide fraction was susceptible to mild alkaline borohydride reduction, yielding a mixture of labeled oligosaccharides which contained N-acetylgalactosaminitol. Thus, the HCT-8R cells are expressing fucosylated mucin-type glycoproteins on their surface.
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PMID:Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas. 405 24

The carbohydrate-binding protein, Concanavalin A (Con A), binds to glucose- or mannose-like sites on the cell-surface membrane. Unless the cells are treated with trypsin, this protein agglutinates malignantly transformed cells, but not normal cells. The transformed cells were agglutinated at 24 degrees C but not at 4 degrees C. Transformed and normal cells treated with trypsin were agglutinated at both 24 degrees C and 4 degrees C with high concentrations of Con A (500 mug/ml), but only at 24 degrees C with low concentrations (5 mug/ml). The same number of Con A molecules were bound to normal and transformed cells at both temperatures. The results indicate that the site for Con A on the surface membrane contains two activities, a component that binds Con A molecules (B) and a component that determines agglutination (A). B is not temperature sensitive and is active in normal and transformed cells, whereas A, which is temperature sensitive, is in an active form only in transformed cells. A can be activated by trypsin, and the increased activity per cell allows agglutination at 4 degrees C with a high, but not with a low, concentration of Con A. Agglutination of transformed cells by wheat-germ agglutinin, which binds to N-acetyl-D-glucosamine-like sites, and by soybean agglutinin, which binds to N-acetyl-D-galactosamine-like sites, was not temperature sensitive. Thus, the temperature-sensitive component A is specific for Con A, and malignant transformation of normal cells, which results in agglutinability by Con A, is associated with the activation of a specific temperature-sensitive activity on the surface membrane.
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PMID:A specific metabolic activity on the surface membrane in malignant cell-transformation. 433 Sep 39

The effect of mild enzyme (trypsin, neuraminidase) treatment, periodate treatment and addition of carbohydrates (mono, di-, and polysaccharides) on the ingestion of Trypanosoma cruzi epimastigotes and trypomastigotes by mouse macrophages was studied. Trypsin treatment did not interfere with the ingestion of epimastigotes but did, however, increase the ingestion of trypomastigotes by mouse peritoneal macrophages. Neuraminidase and periodate treatment of the parasites increased the uptake of epi- and trypomastigote forms. The neuraminidase effect was partially blocked by galactose or N-acetylgalactosamine. Galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine had an influence on the ingestion of T. cruzi by macrophages. This effect was dependent on the strain of parasite tested, and the medium used to cultivate the epimastigotes. The results obtained, in conjunction with the work of others, suggest that glycoproteins and/or glycolipids on the parasite and/or macrophage surface are involved in the T. cruzi-macrophage interaction.
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PMID:Effect of carbohydrates, periodate and enzymes in the process of endocytosis of Trypanosoma cruzi by macrophages. 614 79

Single-cell suspensions of several tumor cell lines, including five human melanomas (A375, SH4, Hs294, Hs852, and Hs939), a human cervical adenocarcinoma (HeLa-S3), a murine melanoma (B16-F1), and a murine fibrosarcoma (UV-2237P), undergo extensive homotypic aggregation in the presence of the glycoproteins fetuin and its desialated derivative, asialofetuin. This phenomenon was observed even at very low glycoprotein concentrations (less than 10 micrograms/ml). Fluorescent derivatives of fetuin and asialofetuin bind to the surface B16-F1 melanoma cells; this binding can be inhibited by lactose (0.1 M). Since the above results suggested the presence of a carbohydrate-binding component(s) on the tumor cells, we tested the possibility that the cells contain endogenous lectin(s). Extracts prepared from the neoplastic cell lines used in this study exhibited a potent capacity to agglutinate trypsin-treated, glutaraldehyde-fixed rabbit erythrocytes. This activity was abolished by treating the extracts with trypsin and could be inhibited by millimolar concentrations of lactose, whereas D-galactose, D-galactosamine, and N-acetyl-D-galactosamine were much less potent inhibitors. D-Mannose, L-fucose, and N-acetyl-D-glucosamine failed to inhibit hemagglutination at 0.2 M. These results demonstrate the presence of a galactoside-specific lectin in the tumor cells. The implications of the existence of a carbohydrate-binding protein(s) on the surface of malignant cells on their in vivo behavior, especially as it may relate to metastatic spread, are discussed.
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PMID:Lectin-like activities associated with human and murine neoplastic cells. 616 52

Inhibition by low-molecular-weight sugars of precipitin line formation between a polysaccharide (EF) excreted by Leishmania tropica subsp. major, Leishmania enriettii, and rabbit antileishmanial antibodies on double gel diffusion plates revealed that galactose residues, possibly as components of lactosyl groups, were the critical immunodominant sugars mediating antibody recognition of EF. The galactose residues of the EF of L. tropica subsp. major were specifically labeled with tritium via galactose oxidase and sodium boro[3H]hydride. The radioactive EF had an apparent molecular weight of about 85,000 on sodium dodecyl sulfate-polyacrylamide gels and was precipitated by antileishmanial antibodies as well as Ricinus communis lectins I and II (galactose specific). Lectins specific for glucose-mannose residues, fucose, N-acetylglucosamine, and N-acetylgalactosamine did not precipitate the labeled EF. Treatment of [3H]EF with proteolytic (trypsin, papain, protease) or glycosidic (alpha-amylase, beta-galactosidase) enzymes had no effect on either the electrophoretic pattern of the material or on its recognition by antileishmanial antibodies or R. communis lectin. This resistance to enzyme activity suggests that EF may be a useful marker for the presence of the parasite in vivo if it can be detected in minute quantities.
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PMID:Identification of galactose as the immunodominant sugar of leishmanial excreted factor and subsequent labeling with galactose oxidase and sodium boro[3H]hydride. 617 74

In the presence of porcine submaxillary N-acetylgalactosaminyltransferase and uridine diphospho-N-acetyl-D-galactosamine, approx. 1.2-1.5 mol of N-acetylgalactosamine were transfered per mol of myelin basic protein. Tritium-labelled N-acetylgalactosamine-labelled basic protein was digested with trypsin and the peptides were separated by HPLC and the radioactivity measured. Most of the radioactivity was associated with three peptide peaks (I, II and III) containing 17, 69 and 6% of the total radioactivity, respectively. The remaining radioactivity was distributed amongst several peptides, each containing less than 2.5% of the total radioactivity. Glycosylation of the basic proteins isolated from human, bovine and guinea pig myelins showed that they were all equally good acceptors. In spite of differences in the peptide profiles of the basic proteins from different species, the distribution of radioactivity between the three peptide peaks was similar for all the species studied. The transfer of N-acetylgalactosamine to peptide II was much faster than to peptides I and III. The apparent Km values of the three peptides were within a narrow range of 0.52-0.63 mM, whereas the Vmax values were considerably different. The glycosylated peptide peaks (I, II and III) were separated by electrophoresis, the radioactivity measured, and amino acid compositions determined after hydrolysis. The major radioactive peptides of the human basic protein were identified with tryptic peptides containing the following sequences: (formula; see text)
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PMID:Identification of the major sites of enzymic glycosylation of myelin basic protein. 619 25

Six peroxidase-labelled lectins have been applied to tissue sections of normal, hyperplastic and lactating human breast. The effect of fixation on binding has been assessed by the comparison of specimens treated with a variety of fixatives with frozen material; the effect of trypsin has also been considered. The other objective has been to establish the consistency of lectin reactivity with this non-malignant breast tissue. Fixation has little effect on the binding of wheat germ agglutinin and peanut lectin, although reactivity with the latter was sometimes increased above that seen in frozen material by the use of Bouin's fluid. Trypsinization of formalin-fixed tissue appears to be a prerequisite for staining with soy bean agglutinin and Ulex europeus. The binding of Lotus tetragonolobus and Dolichos biflorus to fixed tissue has been found to be unreliable in comparison to frozen samples. Soy bean agglutinin and Dolichos biflorus, both specific for N-acetyl-D-galactosamine, have binding patterns which vary between specimens and often between themselves. Of the two fucose-binding lectins Ulex europeus has shown a variability of reaction between specimens, whilst Lotus tetragonolobus consistently reacts with ductal and acinar epithelium but with varying intensities. Consistent binding patterns have been found for wheat germ agglutinin and peanut lectin, the latter after the use of neuraminidase. They therefore form a useful basis for comparison with the reactivity of malignant breast tissue.
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PMID:The binding of peroxidase-labelled lectins to human breast epithelium. I--Normal, hyperplastic and lactating breast. 620

The enzymic transfer of N-acetylgalactosamine to myelin basic protein and that to peptides derived from basic protein were compared. Basic protein treated with pepsin and trypsin before glycosylation resulted in decreases of 7% and 23% in the amount of N-acetylgalactosamine transferred to the peptides respectively. However, digestion of basic protein had little effect on the sites glycosylated. It was found that Thr-95 was the major site for glycosylation in both the intact human basic protein and in the tryptic peptides.
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PMID:The identification of threonine-95 as the major site of glycosylation in normal human myelin basic protein. 620 52


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