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)

Transformation of the chick fibroblast surface has been studied in cells infected with Schmidt-Ruppin Rous sarcoma virus and the temperature-sensitive mutant of this virus, TS-68. Major findings following transformation induced by a shift from nonpermissive (41 C.) to permissive (36 C.) temperature in TS-68 infected cells were: (1) rapid cessation or slowing of the synthesis of a protein, M.W. 100-200,000, localization uncertain; (2) cessation or slowing of the synthesis of a plasma membrane protein, M.W. 45,000, within 2-4 hours; (3) cessation or slowing of the synthesis of a large trypsin- and collagenase- sensitive protein (M.W. greater than 200,000) only after an extended period of morphologic transformation. In addition, increased quantities of type-specific viral antigen in the membranes of infected cells were observed in TS-68-infected cells at 41 compared with 36 C.
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PMID:Comparisons of major cell-surface proteins of normal and transformed cells. 16 7

BALB/c mouse 3T3 cells transformed by simian virus 40 (SV3T3), baby hamster kidney cells transformed by polyoma virus or Rous sarcoma virus, and a range of neoplastic human cell lines release material that inhibits the migration of macrophages and lymphocytes. Similar migration-inhibitory factor (MIF) activity was not detected in supernatants from cultures of untransformed 3T3 or baby hamster kidney cells and a variety of human diploid cell strains. Physico-chemical characterization of the MIF produced by SV3T3 and HeLa cells revealed substantial similarities with the MIF produced by mitogen-activated human peripheral lymphocytes. MIF released by tumor cells is inhibited by pancreatic and soybean trypsin inhibitors and by diisopropylfluorophosphate, indicating that it is a serine-protease. Comparison of MIF produced by SV3T3 cells with a serine-protease plasminogen activator released by the same cells indicated that the latter is more heat labile and has a more heterogenous elution profile after chromatography on Sephadex G-75. The possible role of MIF in causing proteolytic modification of the surface properties of tumor cells and in altering cell-mediated immune responses to neoplastic cells is discussed.
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PMID:Production of a serine-protease with macrophage migration-inhibitory factor activity by virus-transformed cells and human tumor cell lines. 16 63

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.
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PMID:Inhibition of protease activity in cultures of rous sarcoma virus-transformed cells: effect on the transformed phenotype. 16 81

It was previously shown that the fibroblast surface antigen (SF antigen, SFA) is composed of polypeptides of high molecular weight 210,000 (SF210) and 145,000 (SF145) and that both of these decrease in quantity after transformation of the fibroblasts by Rous sarcoma virus (RSV). The present experiments show that SF210 is a glycoprotein. It is accessible to surface labelling by lactoperoxidase catalyzed iodination. The SF210 molecule is highly susceptible to trypsin on cell surface. Anti-SFA antibodies specifically precipitated the surface labelled polypeptide. The lactoperoxidase iodinated SF210 polypeptide was greatly reduced in cells transformed by RSV. It is concluded from these studies that the large external transformation sensitive (LETS) protein detected by other workers is the same molecule as SF210. Part of the label of surface iodinated fibroblasts did not enter the polyacrylamide gels. This high molecular weight material is also susceptible to trypsin treatment and decreases in quantity after transformation by RSV. The data suggest that it may be antigenically related to SF protein. Treatment of surface of 35S-methionine-labelled cultures with trypsin in concentrations able to initiate proliferation of density-inhibited cells rapidly released SF210 from fibroblast surface. A single high molecular weight polypeptide (mol. wt about 200,000, SF200) was detected in the culture medium. SF210 may thus be a major target molecule of trypsin action. Treatment of cultures with insulin that also stimulated the fibroblasts to initiate proliferation did not result in any detectable alteration in the external glycoprotein SF210. It is concluded that although release of SF210 may be a sufficient trigger to stimulate proliferation in stationary cells, this molecule appears not to be directly involved in initiation of fibroblast proliferation from the G1 (or G0) phase of the cell cycle.
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PMID:Fibroblast surface antigen (SF): the external glycoprotein lost in proteolytic stimulation and maligant transfromation. 17 31

Thrombin stimulates cell proliferation in cultures of normal chick embryo fibroblasts but not in cells transformed with Rous sarcoma virus. Analysis of medium conditioned by Rous-sarcoma-virus-transformed cultures demonstrates that these cells do not secrete molecules that can inhibit or inactivate thrombin. The interaction of thrombin with these cells was investigated with enzymatically active 125I-thrombin. The amount of cell-associated 125I-thrombin was found to be three times greater with normal cells than with transformed cells. In both types of cell, greater than 50% of the total cell-associated 125I-thrombin was found as a component that was not dissociated from the cells by trypsin treatment, an observation suggesting that a significant portion was not on the cell surface. The amount of the trypsin-insensitive fraction increases with time up to 12 hr, whereas the trypsin-sensitive fraction is saturated after 1-4 hr. Autoradiography of thin sections of 125I-thrombin-treated cells observed by electron microscopy reveals that after 10 hr incubation greater than 70% of the label is localized in the cytoplasm of both normal and transformed cells. Autoradiograms of sodium dodecyl sulfate/polyacrylamide slab gels demonstrate that 40% of the intracellular label is the size of native thrombin with the remainder in two large fragments of 22,000 and 19,500 daltons.
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PMID:Binding and internalization of thrombin by normal and transformed chick cells. 19 17

Exposure of normal lymphocytes to phytohaemagglutinin or other lectin mitogens results in increased concentrations of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) within minutes. Subsequently, synthesis of purine nucleotides by both the de novo and the salvage pathways is facilitated. This change is prevented by proliferation-inhibiting concentrations of exogenous adenosine. The capacity of lymphocytes to metabolize both adenine and adenosine is increased several-fold by incubation with phytohaemagglutinin but the specific activities of the respective first-step enzymes are not significantly altered. These results suggest that the relatively low quantity of PP-ribose-P available in normal lymphocytes is a major factor limiting the synthesis of purine nucleotides and may be important for the maintenance of the quiescent state. Increased availability of PP-ribose-P may also be associated with proliferative activation of fibroblast-like cells: chick embryo fibroblast cultures released from density-dependent inhibition of growth by insulin, trypsin or serum rapidly increase the rate of adenine incorporation into nucleotides. Chick embryo fibroblasts transformed by Rous sarcoma virus, but not cells infected with the respective non-transforming leukosis virus, show PP-ribose-P concentrations higher than those observed in normal cells.
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PMID:Purine metabolism and control of cell proliferation. 20 61

When microinjected into normal fibroblasts, cytoplasmic extracts of cells transformed by Rous sarcoma virus caused dissolution of microfilament bundles. This activity was not found in extracts of normal cells. The maximum effect was seen within 30 min of injection, and the activity could still be measured after a 10-fold dilution of the cytoplasmic extracts (14 mg/ml original protein concentration). The activity was trypsin sensitive and was destroyed by boiling, but was not RNase sensitive. Protein synthesis was not required for the disruption of actin-containing stress fibers by the injected activity. Microinjected cytoplasts prepared from normal 3T3 cells also showed dissolution of microfilament bundles, indicating that the cell nucleus was not required for expression of activity. Extracts made from fibroblasts transformed by Rous sarcoma virus having a temperature-sensitive mutation in the src gene were also temperature sensitive in the microinjection assay. Thus, the activity of extracts from cells infected with src mutant virus, but not from cells infected with wild-type virus, was destroyed either by in vitro incubation of the extract at the nonpermissive temperature before injection or by incubation of recipient cells at the nonpermissive temperature after injection. We conclude that the microinjection assay can detect a cytoplasmic activity coded for by the src gene of Rous sarcoma virus and that an early direct or indirect target of the src gene product is the cytoskeleton and cell motility system. This result is discussed in relation to the hypothesis that submembranous arrays of microfilaments, microtubules, and their associated proteins interact with cell surface receptors to form a surface modulating assembly that functions as a key regulator of cell growth.
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PMID:Assay for early cytoplasmic effects of the src gene product of Rous sarcoma virus. 20 75

Antibodies to fibronectin and to distinct types of procollagens and collagens were used in immunofluorescent staining to localize these proteins in cell cultures. Normal human skin or lung fibroblasts produced a fibrillar pericellular matrix in which fibronectin and procollagen (types I and III) showed extensive codistribution. Fibronectin and procollagen were synthesized by the same cells as judged by double-stain immunofluorescence. Pericellular procollagen was specifically digested with collagenase without an effect on the fibrillar distribution of matrix fibronectin. Brief treatment with trypsin removed both matrix proteins. The human tumor cell lines HT-1080 (fibrosarcoma) and RD (rhabdomyosarcoma) produced little or no matrix fibronectin or procollagen. At sites of cell contact, simian virus 40-transformed lung fibroblasts (VA13) produced small amounts of pericellular fibrillar matrix fibronectin that codistributed with procollagen type I. Intracellular fibronectin and procollagen were visualized in all of these human sarcoma cell lines. When chicken embryo fibroblasts infected with a T class mutant (NY68) of Rous sarcoma virus temperature-sensitive for transformation were maintained at the nonpermissive temperature (41 degrees ) the cells had normal phenotype and a fibrillar matrix containing fibronectin and procollagen was present. At the permissive temperature (35 degrees ), the cells showed transformed phenotype and the matrix was lost. The failure to produce a pericellular fibronectin/collagen matrix may account for several phenotypic characteristics of transformed cultured fibroblasts.
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PMID:Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen. 21 6

Comparisons of membrane glycopeptides from baby hamster kidney fibroblasts (BHK21/C13) and a clone transformed by Rous sarcoma virus (C13/B4) were made by using cells metabolically labeled with radioactive D-glucose and L-fucose. Most of the glycopeptides were metabolically labeled with both the general and the specific glycoprotein precursors. The glycopeptides obtained from the cell surface by controlled trypsinization were representative of the surface membrane as shown by comparing them with those of purified membrane preparations. The trypsin-removable glycopeptides from both cell types were further processed and examined by successive chromatography on Sephadex G-50 and DEAE-cellulose. The chromatographic distribution patterns showed that each cell type had glycopeptides of similar characteristics, although the proportions of the glycopeptides differed dramatically between the two cell types. After transformation there was an increase in the larger, more highly charged glycopeptides. This was verified by the increased sialic acid content in these glycopeptides. Some of the glycopeptides were homogeneous after the size and charge separations, since a variety of procedures did not separate them further. The apparent homogeneity and reasonably few species obtained may be due to the methods of isolation, with the procedures selecting particular glycopeptides from the external portion of the membrane. These results corroborate the concept and show for the first time that virus transformation is accompanied by an increase in certain species of glycopeptides rather than de novo synthesis.
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PMID:Membrane glycopeptides from virus-transformed hamster fibroblasts and the normal counterpart. 22 Oct 10

The predominant surface glycopeptide from a clone of baby hamster kidney cells transformed by Rous sarcoma virus (C13/B4), metabolically labeled with L-[14C]fucose, has been characterized for the first time. This glycopeptide represents 19% of the total radioactivity removed by trypsin from the cell surface of the transformed fibroblasts and is more abundant in the transformed cells than in the normal counterpart. Purification of the glycopeptide after digestion with Pronase was by successive chromatography on DEAE-cellulose and Sephadex G-50. The monosaccharide content of the glycopeptide was 42, 127, 138, 114, and 243 nmol of fucose, sialic acid, galatose, mannose, and glucosamine, respectively. A partial structure of the glycopeptide was proposed from the results of sequential enzymatic degradation coupled with gas-liquid chromatographic analysis of the resultant monosaccharides. All of the enzymes used were purified and pretested on natural substrates and found to remove terminal monosaccharides of the correct configuration, quantitatively. The purification and properties of an alpha-L-fucosidase from rat testes were described. All of the radioactivity in the glycopeptide, recovered as fucose, was present at the core and was removed by treatment with this alpha-L-fucosidase. The proposed structure is a triantennary, completely sialylated, complex glycopeptide containing a core region of beta-D-mannose, beta-D-N-acetylglucosamine, and alpha-L-fucose.
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PMID:Partial structure of a membrane glycopeptide from virus-transformed hamster cells. 22 Oct 11


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