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)

The mobility of plant lectin receptors in the plane of the membrane is examined for cells prepared from embryonic chick neural retinas by a variety of procedures. Cells liberated from the intact tissue by trypsin treatment followed by mechanical dissociation are able to redistribute their receptors into 'caps' both spontaneously and in the presence of a multivalent lectin. These cells, dispersed by trypsinization, upon repair in culture for a suitable period of time lose their ability to redistribute lectin receptors. Cells dispersed by mechanical means without prior trypsin treatment are unable to undergo 'cap' formation. In addition, cells within intact tissues are also unable to redistribute their lectin receptors into 'caps.' Based on these observations we propose that within solid tissues which have assumed their characteristic architecture, cell surfaces are immobilized, and that this phenomenon may be a critical parameter in determining the potential of a cell to undergo morphogenetic rearrangements.
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PMID:Spontaneous and lectin-induced redistribution of cell surface receptors on embryonic chick neural retina cells. 123 8

A major cell-surface glycoprotein of the TA3-Ha ascites mammary adenocarcinoma diminished during transfer from ascites growth to cell growth in suspension culture. A sensitive, hemagglutination-inhibition assay that used a lectin from Vicia graminea seeds indicated approximately a 50% loss after 7-10 days of culture and a 90% loss after 2 months. These findings were corroborated by carbohydrate and amino acid analysis with gas-liquid chromatography of trypsin glycopeptides released from the cell surface. Repassage of the cultured cells in vivo caused the reappearance of the surface glycoprotein.
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PMID:Reversible loss in suspension culture of a major cell-surface glycoprotein of the TA3-Ha mouse tumor. 123 14

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. alpha-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B. Resealed membranes preparaed with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes. Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that shown no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37 degrees C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present. Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP. The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells in different biological states, such as those encountered in normal and transformed cells.
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PMID:Effect of metabolic state on agglutination of human erythrocytes by concanavalin A. 124 21

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.
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PMID:The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C. 126 13

Glycopeptide-containing fractions in HPLC peptide maps can be detected by a simple application of the microtiter plate-bound streptavidin-biotinylated glycopeptide-lectin method (M.-C. Shao, 1992, Anal. Biochem., 205, 77-82). To illustrate this application, the glycoproteins, ovalbumin and asialofetuin, reduced and S-alkylated with vinylpyridine, were digested with trypsin-L-1-p-tosylamino-2-phenylethylchloromethyl ketone and the tryptic peptides were fractionated by reverse-phase HPLC, monitoring for absorbance at 230 nm. Aliquots of the HPLC fractions (typically 0.2-0.5% of the total volume) were biotinylated and complexed with streptavidin in the wells of a microtiter plate, allowing the streptavidin-glycopeptide complex to adhere to the plate. Suitable lectins, such as concanavalin A, Datura stramonium agglutinin, and peanut agglutinin, all of which had been coupled to horse radish peroxidase, were added, and after thorough washing, only the wells containing streptavidin-bound glycopeptides retained the complementary lectin and gave a positive peroxidase reaction. Less than 1 pmol of glycopeptide can be detected. The demonstration that the glycopeptide detection could be inhibited either by addition of an excess of the appropriate sugar inhibitor to the different lectins or by digestion of the biotinylated glycopeptides with N-glycosidase F or O-glycosidase shows that the glycopeptide-lectin interaction is the basis for the reaction.
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PMID:Method for the detection of glycopeptides at the picomole level in HPLC peptide maps. 128 90

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.
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PMID:The human placental transferrin receptor: reconstitution into liposomes and electron microscopy. 129 37

We have tested the hypothesis that some phenotypic characteristics of the lymphocytes from mice with lymphoproliferative disease (lpr) could be explained by abnormal glycosylation of membrane proteins. Lymph node cells from normal C57 BL/6 and from C57 BL/lpr mice were labelled with tritiated sugars. Membrane proteins were released with trypsin, then with pronase. After complete pronase digestion, glycopeptides were first separated on Bio Gel P-6 and then on Con A-Sepharose. Fractions not binding to Con A (Con A negative) were also separated on Lens culinaris agglutinin-Sepharose. Marked differences between normal and lpr cells were noticed. First, there were more glucosamine-labelled peptides with very high molecular weight (eluting fast on Bio Gel P-6) on lpr cells than on normal lymphocytes. Second, the proportion of mannose-labelled peptides binding to Con A was smaller in the lpr cells. Third, among the Con A negative peptides, the proportion binding to Lens culinaris agglutinin was higher in lpr cells. Thus, lpr cells seem to carry more alpha 1-6 fucosylated chains and larger size carbohydrates. These alterations were also confirmed by gel electrophoresis of lectin-selected iodinated cell surface antigens and seem to be restricted to a very limited number of peptides. Thus, there may be primary changes in glycosylation in lpr cells. Alternatively, the glycosylation pattern of lpr cells may be characteristic for a subpopulation of T-lymphocytes that is expanded in this disease, or for a certain stage of activation. A large proportion of Con A-negative, Lens culinaris-positive peptides is a rather unusual feature in murine cells and requires further investigation.
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PMID:Comparison of the N- and O-linked glycopeptides of lymph node cells from C57 BL/6 lpr/lpr and C57 BL/6 mice. 129 47

Several high-density lipoprotein (HDL)-binding proteins, candidates for the putative HDL receptor, have recently been identified, including two membrane proteins: HB1 of 120 kDa and HB2 of 100 kDa, present in rat and human liver plasma membranes respectively. Further insights into their function however, have been hampered by poor recoveries of these hydrophobic peptides, and the present work was undertaken to improve yields and enable a more detailed investigation of their properties. A significant improvement has been achieved using two affinity chromatographic procedures, one exploiting the glycoprotein nature of the proteins and the other exploiting their ligand properties, which in combination resulted in considerable enrichment of HB1 and HB2. Thus DEAE-Sephacel fractionation (0.05-0.2 M-NaCl) of CHAPS-solubilized plasma membranes yielded active HDL-binding proteins which bound to concanavalin A-Sepharose or wheat-germ-lectin-Sepharose columns and retained their binding activity after eluting with methyl-alpha-D-mannoside or N-acetylglucosamine respectively. These glycoproteins were further purified by affinity chromatography using apo-HDL-Sepharose columns. Final purification required preparative SDS/PAGE. Investigation of the carbohydrate moieties of the proteins using glycosidases and two-dimensional gel electrophoresis revealed pI values ranging from 4.6 to 4.9 and from 4.5 to 4.7 for HB1 and HB2 respectively, which after treatment with neuraminidase shifted towards basic pH (5.4-5.7 and 5.3-5.5 respectively). The molecular masses were decreased to 115 kDa and 95 kDa respectively, demonstrating that sialic acid residues contributed significantly to the negative charge of the glycosylated peptides. Treatment with the enzyme peptide N-glycosidase F (N-glycanase) resulted in a decrease in molecular mass of HB1 and HB2 to 105 kDa and 80 kDa respectively, but endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment was not effective. Neither neuraminidase nor N-glycanase treatment destroyed activity, suggesting that sialic acids or N-linked oligosaccharides are not important determinants of HDL binding. Digestion of plasma membranes with trypsin or Pronase resulted in a loss of activity of both HB1 and HB2 that was not influenced by prior treatment with neuraminidase, suggesting that sialic acid residues play no protective role against proteolytic cleavage of HDL receptor proteins.
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PMID:Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties. 131 18

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.
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PMID:Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity. 133 45

Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of 125I labelled Evonymus europaea and Griffonia simplicifolia I-B4 lectins. Treatment of the stimulated macrophages with coffee bean alpha-galactosidase abolished binding of the GS I-B4 isolectin and changed the binding pattern of the Evonymus lectin. The affinity (Ka) of Evonymus lectin for alpha-galactosidase-treated macrophages decreased approximately 23-fold, from 1.25 x 10(8) M-1 to 5.5 x 10(6) M-1. Subsequent digestion of alpha-galactosidase-treated macrophages with alpha-L-fucosidase from Trichomonas foetus, further reduced binding of Evonymus lectin. Resident macrophages showed the same pattern of Evonymus lectin binding, with the same affinity, as alpha-galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of the Evonymus lectin which, in the absence of alpha-D-galactosyl groups, requires alpha-L-fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal alpha-L-fucosyl residues. It is also concluded that during macrophage stimulation/activation alpha-D-galactosyl residues are added to this glycoconjugate and that they form part of the receptor for Evonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains alpha-D-galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound to Corynaebacterium parvum activated macrophages.
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PMID:Alpha-D-galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages. 134 14


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