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 orientation of proteins and glycoproteins of the platelet surface has been studied using various surface probes and labeling reagents. A fourth major glycoprotein has now been detected in platelet plasma membranes by sodium dodecyl sulfate-gel electrophoresis in addition to the previously recognized glycoproteins I, II, and III. Glycoprotein IV Mr, = approximately 87,000) appears to be present on the inner aspect of the membrane or buried within it since it is not accessible to surface probes such as lactoperoxidase-catalyzed iodination, radiolabeling with transglutaminase and [14C]glycine ethyl ester, or proteolytic enzymes. The ratio of these four major membrane-bound glycoproteins is approximately 10:4:2:3. Contrary to previous reports, only one glycoprotein, glycoprotein III, is accessible to lactoperoxidase-catalyzed iodination in intact platelets. Differences in the rate of destruction of glycoprotein II in intact platelets by trypsin suggests that two components may be migrating in this region. Examination of the soluble fraction obtained following platelet homogenization showed the presence of a single soluble glycoprotein of molecular weight 148,000 comprising about 10% of total platelet sialic acid. Treatment of intact platelets with neuraminidase resulted in the quantitative loss of siliac acid from the soluble glycoprotein, and it was strongly labeled in the intact platelet by [14C]glycine ethyl ester in the presence of transglutaminase. Treatment of intact platelets with chymotrypsin which does not cause the platelet release reaction, caused the rapid conversion of the soluble glycoprotein to a macroglycopeptide. These results indicate a surface origin for the soluble glycoprotein rather than a cytoplasmic or granular origin. The term glycocalicin is suggested for this glycoprotein in view of its origin in the platelet glycocalyx.
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PMID:Platelet glycocalicin. I. Orientation of glycoproteins of the human platelet surface. 82 54

The topography of the external surface of the human red cell membrane has been studied using an impermeant radioactive probe, [125I]diazodiiodosulfanilic acid, which binds covalently to protein groups of the membrane following reaction with intact cells. The pattern of labeling was assessed by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis followed by sequential analysis of single gels for carbohydrates (by staining with the periodic acid-Schiff (PAS) reagent), for proteins (by staining with Coomassie blue), and for radioactivity (by counting gels sliced in 2-mm segments). The radioactive probe bound to membrane polypeptides with apparent molecular weights of 94,200, 58,100, and 46,500 (Peaks A, B, and C, respectively). Peak A co-migrated with a small periodic acid-Schiff-positive band and protein Band 3 (nomenclature of Steck) (Steck, T.L. (1974)J. Cell Biol. 62: 1-19). Peak B migrated with protein Band(s) 4.5 slightly ahead of the major membrane glycoprotein (PAS-1). Peak C migrated like glycoprotein PAS-2 and protein Band 5, the actin-like, water-soluble membrane protein. In contrast to lactoperoxidase iodination and a number of other probes, [125I]diazodiiodosulfanilic acid reacted minimally with the major membrane glycoprotein, glycophorin. When it was reacted with isolated ghosts, all molecular weight classes of polypeptides were labeled. Treatment of labeled cells with neuraminidase or trypsin altered the glycoprotein staining pattern, but not the radioactive peaks. On the other hand, Pronase eliminated the Mr=94,200 radioactive peak, diminished the other two radioactive peaks, and profoundly changed the glycoprotein and protein staining patterns. Treatment of the membranes of labeled cells in a low ionic strength alkaline medium did not alter radioactive peaks and demonstrated that Peak C differed from the actin-like membrane protein. A nonionic detergent, Triton X-100, solubilized all radioactive components. The studies have defined the binding of [125I]diazodiiodosulfanilic acid to external proteins of the human red cell membrane. Its pattern of reaction differs quantitatively and qualitatively from other commonly used reagents, and it provides a useful additional vectorial probe for the study of membrane topography. Its reactions provide further evidence of the organizational complexity of the red cell membrane and emphasize the fact that interpretation of information derived from the use of membrane probes must take into account the differences resulting from the properties of the probing reagents themselves.
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PMID:Topography of the external surface of the human red blood cell membrane studied with a nonpenetrating label, [125I]diazodiiodosulfanilic acid. 83 50

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of 'ghosts' radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.
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PMID:Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane. 85 15

We have developed a new method for identifying proteins which span the plasma membrane ("trans-membrane" proteins) of mammalian cells grown in tissue culture. The method involves labeling proteins exposed on the cell surface with 125I by the lactoperoxidase technique and then preparing sealed, "inside-out" membrane vesicles (phagosomes) from the labeled cells using the polystyrene latex bead procedure. These inside-out vesicles are then treated briefly with trypsin and analyzed by SDS-polyacrylamide gel electrophoresis for the presence of 125I-labeled protein species which were degraded by proteolytic attack. Such proteins must be exposed on both the outer and inner membrane surfaces and, therefore, they must pass through the lipid barrier. This method is a general one in the sense that it is suitable for use with a wide variety of cell types, and here we show how it has been employed to prove that a particular high molecular weight polypeptide, called band 1, spans the plasma membrane of mouse L cells. Further studies of the band 1 polypeptide have demonstrated that it is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in the availability of band 1 to iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of cell proliferation.
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PMID:Identification of a high molecular weight trans-membrane protein in mouse L cells. 103 Aug

The structure of the major human erythrocyte membrane protein (protein E) was investigated by studying the products of proteolysis of the native protein in the membrane. The distribution and location of the tyrosine residues labelled by radioiodination by lactoperoxidase was determined. Proteolysis of the extracellular region of the protein by thermolysin released four tyrosine-containing peptides, all of which were also found to remain in the major fragment that is retained in the membrane. The presence of these duplicated sites in the extracellular region of the protein was confirmed by limited trypsin digestion of the intracellular region of the protein. Two groups of fragments were obtained. Both groups contained a set of the extracellular labelled sites, but they differed in containing distinct groups of intracellular sites, showing that the two sets of extracellular sites are linked by an intracellular region of the protein. The polypeptide chain thus traverses the membrane twice. An S-shaped model which is consistent with these data is proposed.
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PMID:The major human erythrocyte membrane protein. Evidence for an S-shaped structure which traverses the membrane twice and contains a duplicated set of sites. 116 51

Milk fat globule membrane was solubilized with sodium dodecyl sulfate and mercaptoethanol and the membrane proteins were separated by SDS-polyacrylamide gel electrophoresis. The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons. At least five glycopeptides were separated of which two stained intensely with periodic acid-Schiff reagent, but poorly with coomassie blue. Trypsin hydrolysis of whole cream and isolated milk fat globule membrane revealed major differences in the rates of protein hydrolysis. Many of the membrane proteins of whole cream resisted proteolysis compared with the same proteins in the isolated membrane. Two glycopeptides were resistant to trypsin digestion in either preparation. Treatment of whole cream with neuraminidase led to the release of at least 70% of the protein-bound sialic acid. Whole cream and isolated membrane samples were iodinated with 125I in the presence of lactoperoxidase and hydrogen peroxide. The membrane proteins were significantly more accessible to lactoperoxidase-125I i in isolated membrane compared with the proteins of whole cream. Polypeptides of molecular weight 43,500 and approximately 48,000 daltons were predominantly labelled in whole cream and could be eluted from the fat globules with magnesium chloride (1.5m). The results strongly suggest that the proteins of milk fat globule membrane are asymmetrically arranged in the membrane and that most of the protein-bound sialic acid is present on the external surface of milk fat globules.
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PMID:Studies on the structure of milk fat globule membrane. 119 40

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.
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PMID:Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures. 125 8

A genomic library of Mycoplasma hyopneumoniae was constructed by cloning random DNA fragments approximately 300 base pairs long in a fusion expression plasmid, pEx29, containing the N terminus of the phage MS2 polymerase under the control of the PL promoter of phage lambda. Clones that produced fusion proteins carrying surface-specific antigenic determinants were identified by using antiserum raised in a pig by intranasal inoculation of viable mycoplasmas. Rabbit antisera produced against gel-purified fusion proteins synthesized in Escherichia coli were analyzed by Western blotting to identify antigenically related mycoplasma components. Distinct mycoplasma proteins termed P90, P68, P50, P30, and P26 were identified. Evidence for the surface location of P90, P68, and P50 was provided by their sensitivity to trypsin and their comigration with lactoperoxidase-catalyzed iodinated proteins of intact mycoplasmas. Immune electron microscopy, performed with antiserum against the hybrid MS2-mycoplasma protein produced in E. coli and corresponding to P90, also showed that its antigenic determinant is associated with the mycoplasma surface.
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PMID:Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library. 241 Mar 63

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.
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PMID:Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae. 241 45

Cytoplasmically exposed portions of the high affinity receptor for immunoglobulin E were investigated with controlled proteolytic digestion of plasma membrane vesicles from rat basophilic leukemia cells. Hypotonic shock treatment results in vesicle inversion, thereby exposing the cytoplasmic portions of the approximately 32 kDa beta and approximately 8 kDa gamma subunits to surface labeling by lactoperoxidase-catalyzed 125I-iodination. These 125I-labeled protein segments disappeared after treating inverted vesicles with trypsin, and labeled components also disappeared when chloramine T mediated 125I-iodination was used to label receptors after inverted vesicles had been trypsin digested and solubilized. Biosynthetic labeling of receptors with 35S-methionine showed that a 17-19 kDa labeled fragment, designated beta', remains associated with alpha after trypsin digestion of inverted vesicles. This beta' fragment was confirmed to be the intramembranous portion of the beta subunit in experiments where receptors were labeled with the hydrophobic photoactivated probe 3-(trifluoro-methyl)-3-(m-[125I] iodophenyl)diazirine prior to digestion. Our experimental results are consistent with the amino acid sequence and topography of the beta subunit predicted from the recently cloned cDNA for this subunit (Kinet, J.-P., et al., Proc. Natl. Acad. Sci. 85, 6483, 1988). Since the cytoplasmically-exposed portions of the beta and gamma subunits can also be efficiently and selectively removed from solubilized receptors by trypsin, reconstitution experiments to examine the importance of these segments in mediating the delivery of the transmembrane signal are made possible.
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PMID:Proteolytic digestion of the beta and gamma subunits of the receptor for immunoglobulin E at the cytoplasmic face of the plasma membrane. 252 81


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