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)

Purified human plasma fibronectin at concentrations of about 30 microgram/ml was found to agglutinate trypsin-treated erythrocytes from certain species. The hemagglutination reaction was inhibited by specific antibodies to fibronectin, by relatively low concentrations of polyamines and by higher concentrations of basic amino acids and nonacetylated amino sugars. The divalent cations Ca2+ and Mg2+ and the chelating agent ethylenediaminetetraacetate did not affect the reaction. None of the neutral amino acids, neutral sugars or polyanions tested was inhibitory. The results imply that plasma fibronectin is capable of interacting with cell surfaces and support the idea of a similarity between cellular and plasma fibronectins.
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PMID:Hemagglutinin activity of human plasma fibronectin. 11 52

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-(3)H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated (3)H was present as [(3)H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [(3)H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [(3)H]fucose-labelled material before and after trypsin digestion. 3. The [(3)H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000-250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [(3)H]fucose and (14)C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [(35)S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.
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PMID:Biosynthesis and release of glycoproteins by human skin fibroblasts in culture. 20 58

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

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

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.
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PMID:Cross-linking of fibronectin to sulfated proteoglycans at the cell surface. 22 72

Cells growing on plastic or glass surfaces in vitro may be brought into suspension by proteases (e.g. trypsin) or chelating agents (e.g. EGTA). Trypsin and EGTA remove different quantities and types of molecules from cell surfaces. Previous studies have revealed that when confluent cultures of either BHK or PyBHK cells are brought into suspension by exposure to trypsin, foetal calf serum (or fibronectin) is required for cell attachment to films of denature type I collagen, but not to 3-dimensional gels of native collagen fibres. In this communication the serum requirements for the attachment of BHK and PyBHK cells to collagen substrata have been examined as a function of (a) the method used to prepare the cell suspension (EGTA or trypsin), and (b) cell density. Data are presented consistent with the view that cell surface-associated fibronectin is able to mediate cell attachment directly to films of denatured collagen.
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PMID:The effects of EGTA and trypsin on the serum requirements for cell attachment to collagens. 23 8

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.
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PMID:Fibronectin and proteoglycans as determinants of cell-substratum adhesion. 23 21

The formation of a highly organized vascular and corneal endothelial cell monolayer is associated with the appearance of a 60,000-dalton cell surface protein (CSP-60) (30,000 daltons after reduction with dithiothreitol) which is not detectable in rapidly growing endothelial cells and in subconfluent cultures that do not yet exhibit the strict morphology of a confluent monolayer. It is also absent from vascular smooth muscle cells and from endothelial cultures that are maintained in the absence of fibroblast growth factor and grow on top of each other at confluence. After disorganization of cells in a confluent endothelial monolayer by urea, EDTA, or trypsin, CPS-60 is no longer exposed on the cell surface, but it reappears as soon as the cells readopt their characteristic two-dimensional configuration. This reorganization can be achieved in the presence of cycloheximide and despite removal of fibronectin by urea, EDTA, or trypsin. Maximal amounts of fibronectin and no CSP-60 are detected in subconfluent, but not yet organized, endothelial cultures or in endothelial cells that no longer form a monolayer of nonoverlapping cells at confluence. Likewise, cultures of vascular smooth muscle cells contain fibronectin but no CSP-60. These results suggest that CSP-60, rather than fibronectin, could be involved in the adoption of a monolayer configuration by confluent endothelial cells.
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PMID:Appearance in confluent vascular endothelial cell monolayers of a specific cell surface protein (CSP-60) not detected in actively growing endothelial cells or in cell types growing in multiple layers. 28 73

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.
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PMID:Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix. 36 26

Soluble 125I-labeled type I collagen binds to cultured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences contained within the helical portions of the alpha1 and alpha2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Binding is decreased but measurable at 4 degrees C. The steady state binding is greater at 26 degrees than at 37 degrees C due to a more rapid dissociation of the ligand-acceptor complex at 37 degrees C. The half-life of the complex is 46 min at 37 degrees C and approximately 2.5 hr at 26 degrees C. Scatchard plots of binding data indicate a single class of high affinity binding sites (KD = 1.2 X 10(-11) M) with each fibroblast binding approximately 500,000 molecules at saturation. Pretreatment of fibroblasts with bacterial collagenase, chondroitinase ABC or testicular hyaluronidase does not affect the binding reaction, whereas pretreatment of the cells with phospholipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concentrations which remove surface fibronectin. Fibroblast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibronectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.
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PMID:Binding of soluble type I collagen molecules to the fibroblast plasma membrane. 45 36


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