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 specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.
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PMID:Proteins of the hepatoma tissue culture cell plasma membrane. 0 57

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.
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PMID:Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid. 0 17

Sera from chickens naturally infected with Marek's disease herpesvirus (MDHV) form preciptin lines with at least two immunologically distinct soluble antigens designated MDHV-A and MDHV-B. Partial purification and characterization of the glycoprotein MDHV-A antigen was previously reported. MDHV-B was found predominantly in the sonically treated extracts of infected cells, in contrast to the predominantly extracellular MDHV-A. Analysis of these extracts from [14C]glucosamine-labeled cells by immunodiffusion with chicken anti MDHV-B serum negative for MDHV-A followed by autoradiography confirmed that MDHV-B was a common antigen between MDHV and herpesvirus of turkeys and revealed that it was also a glycoprotein. Because of their glycoprotein nature, both MDHV-A and MDHV-B bound to concanavalin A affinity chromatography columns and could then be eluted by alpha-methyl-D-mannoside and recovered for further analysis. Concanavalin A affinity chromatography was an excellent technique for initial purification of MDHV-A and MDHV-B, since approximately 5- and 15- fold purification, respectively, was achieved in a single simple step. MDHV-B was resistant to trypsin under conditions where MDHV-A was sensitive, but was similar to MDHV-A in resistance to pH 2.0 and to 1.0 or 2.0 M urea and 0.05% Brij 35. Partially purified MDHV-B was analyzed by sucrose gradient sedimentation, isoelectric focusing, and gel filtration on Sephadex G-200 in the presence of 1.0 or 2.0 M urea and 0.05% Brij 35 to purify the antigen and to determine its physical and chemical properties in comparison with those already reported for MDHV-A. MDHV-B had a much lower isoelectric point in pH 4,54, a higher sedimentation coefficient of 4.4S, and a greater molecular weight of 58,250. These data indicate that MDHV-B is physically distinct from MDHV-A antigen, although the size difference is not sufficient to allow for effective separation. In contrast, the isoelectric point difference of greater than 2 pH units makes isoelectric focusing an effective means of purifying the antigens free of one another. The four-step purification procedure achieved greater than 200-fold purification of MDHV-B. Immunization of rabbits with this highly purified antigen results in the preparation of antisera that appeared monospecific for MDHV-B in immunodiffusion.
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PMID:Marke's disease herpesviruses. III. Purification and characterization of Marek's disease herpesvirus B antigen. 2 34

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.
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PMID:Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides. 5 39

Three immunologically distinct types of polysaccharides have been isolated by diethylaminoethyl (DEAE) chromatography from the lipopolysaccharide extracts of group B Neisseria meningitidis. All types contain a set of common determinants, as well as distinct ones; all of these determinants are detectable by either immunodiffusion or enzyme-linked immunosorbent assay (ELISA). The polysaccharides elute from a Sepharose 4B column in the range of 2-3 x 10(5) daltons and have isoelectric points from 4.2 to 4.3. Their antigenicity is destroyed by oxidation but is unaffected by neuraminidase, lysozyme, or trypsin. One type of polysaccharide cross-reacts with the Gc2 polysaccharide of Neisseria gonorrhoeae in immunodiffusion systems. Chemical analysis indicates that these polysaccharides contain hexoses, hexosamines, 2-keto-3-deoxyoctonate, ethanolamine, and heptose; analysis of amino acids indicates protein contents of less than 0.05%. In contrast to the lipopolysaccharide from which they are derived, these polysaccharides contain no lipid A and less than 0.5% fatty acids. All three types are precipitated by wheat germ agglutinin but not by concanavalin A or fucose-binding protein. Specific inhibition of this precipitation can be achieved with N-acetyl glucosamine. These antigens may be the bases of a lipopolysaccharide-derived typing system for group B N. meningitidis.
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PMID:Lipopolysaccharide-derived serotype polysaccharides from Neisseria meningitidis group B. 8 91

A preparation rich in basement membranes isolated from rat testes (STBM) was exposed to pepsin, collagenase, trypsin, and pronase to obtain soluble fractions. The immunological reactivity of these fractions was studied by gel immunodiffusion or by passive hemagglutination tests against an anti-STBM serum. All fractions reacted with the antiserum, but the highest titer was detected when the antiserum was reacted with a fraction that contained only traces of hydroxyproline (fraction 1), whereas low titers were obtained with collagen or collagen fragments isolated from STBM. Antibodies in the anti-STBM serum were mainly directed to the glycoproteins of STBM not related to collagen. Fraction 1, obtained by subsequent collagenase and trypsin digestion of STBM and purification by Sephadex G-200, was a high molecular weight glycoprotein that was free of half-cystine and methionine, had only traces of hydroxyproline, and contained 7.2% neutral sugars, 0.26% sialic acid, and 8.7 residues of glucosamine per 1000 residues of amino acids.
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PMID:Isolation and immunological reactivity of soluble fractions from rat seminiferous tubule basement membrane. 9 Apr 90

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.
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PMID:Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture. 12 54

A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.
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PMID:Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells. 16 3

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cells cultured at the permissive (32 degrees C) and non-permissive (39 degrees C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3-4 generations in medium containing radioactive leucine (32 degrees C and 39 degrees C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (congruent to 100 000-160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growth (39 degrees C). A decrease in amount of a 120 000 molecular weight glycopeptide at 39 degrees C was the most prominent of these alterations. We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 degrees C, using lactoperoxidase-catalyzed iodination, NaBH4 reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.
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PMID:Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties. 17 78

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.
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PMID:Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells. 17 96


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