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)

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 chondrocyte is a specialized cell that synthesizes proteoglycans of a type found only in cartilage and nucleus pulposus. These proteoglycans are distinct in forming multiple aggregates of unique structure in which hyaluronic acid provides a central chain to which many proteoglycan molecules are bound at one end only. Chondrocytes were isolated from adult cartilage and used in suspension culture to test the effect of compounds in the medium on the synthesis of proteoglycans. Hyaluronic acid alone, among a number of compounds extracted from or analogous to those in cartilage, reduced the incorporation of [35S] sulphate into macromolecular material. Oligosaccharides of hyaluronic acid of the size of decasaccharides and above also had this effect but hyaluronic acid already bound to proteoglycan did not. The proportion of total labelled material associated with the cells increased at the expense of that in the medium. Treatment of the cells with trypsin abolished the effect of hyaluronic acid but treatment with chondroitinase did not. It is suggested that hyaluronic acid interacts with proteoglycans at the cell surface by a specific mechanism similar to that involved in proteoglycan aggregation, as a result of which the secretion and synthesis of proteoglycans is reduced.
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PMID:Influence of the cells on the pericellular environment. The effect of hyaluronic acid on proteoglycan synthesis and secretion by chondrocytes of adult cartilage. 23 23

Human polymorphonuclear neutrophil (PMN) granule extract (25 mug of protein) released 60 percent of the available 35SO4 from labeled rabbit articular cartilage in 0.5 hour at neutral pH. N-acetyl-L-alanyl-L-alanyl-L-prolyl-L-alanine choloromethyl ketone (NAcAAPACK), a specific elastase inhibitor, was only minimally effective against whole granule extract, and N-alpha-tosyl-L-lysine chloromethyl ketone, which inhibits trypsin but not elastase, was completely ineffective. Preparative disc-gel electrophoresis of PMN granule extract revealed two separate regions with independent activity against 35SO4-labeled cartilage. One region contained elastases and when tested alone, was completely inhibited by NAcAAPACK. The other contained lysozyme and two esterases active against N-acetyl-L-phenylalanine-alpha-naphthol. Purified lysozyme proved inactive, suggesting that the chymotrypsin-like esterases were responsible for proteoglycan degradation by this region of the gel.
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PMID:Identification of neutral proteases in human neutrophil granules that degrade articular cartilage proteoglycan. 23 25

The distribution of glycosaminoglycans and glycoproteins has been studied in cytoplasmic and particulate fractions of neurons isolated in bulk from rat cerebrum. Lysis of the neurons in 25 mM sodium phosphate buffer at pH 7.5 released 20% of the protein and over 90% of the lactate dehydrogenase in a soluble form. Eighty-two percent of the chondroitin sulfate was also released, together with 55% of the heparan sulfate and 24-25% of the hyaluronic acid and glycoproteins. The chondroitin sulfate remaining in the membranes was completely depolymerized to disaccharides after treatment with chondroitinase ABC, and treatment of the neuronal membranes with 0.1% trypsin removed 55-63% of the chondroitin sulfate and heparan sulfate but only 25% of the sulfated glycoproteins. The results reported here support our previous conclusion that the soluble chondroitin sulfate proteoglycan of brain is largely a cytoplasmic constitutent of neurons (and astrocytes) and is not primarily present in nervous tissue as an extracellular ground substance.
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PMID:Presence of chondroitin sulfate in the neuronal cytoplasm. 28 11

The state of chick embryo chondroblasts in culture was found to be sensitive to both fibronectin and another substance(s) (activity A) which could be extracted from chick embryo fibroblasts with 1 M urea or from conditioned medium. In the presence of either of these activities at concentrations of 25-150 micrograms/ml, chondroblasts, which normally grow as mixed cultures of floating and adherent cells, all immediately became attached to the tissue culture dish and spread. After several days, the morphology of these typically epithelioid cells became fibroblastic. This did not involve a selection process, since the effect was reversible. The synthetic program of these cells was also dramatically modified: the cultures no longer synthesized the chondroblast-unique type IV sulfated proteoglycan and began synthesizing alpha 2 collagen chains typical of fibroblastic or early limb bud cells. Fibronectin was resolved from activity A by gelatin affinity chromatography or gel filtration. Both activities were trypsin-sensitive. The two activities differed, however, on the basis of how the protein fractions in which they were found migrated in SDS-polyacrylamide gels, their specific activities and their effects on cell morphology and cell growth.
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PMID:Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts. 47 27

Partially degraded hyaluronate was coupled to AH-Sepharose 4B using carbodiimide. Approximately 1 mg of hyaluronate was incorporated per ml of wet gel. The derivatized gel was used to purify components of the hyaluronate-proteoglycan complex of cartilage. Two link-proteins were isolated from a crude cartilage extract by affinity binding to the gel and eluted with 4 M guanidinium chloride. By the same procedure one link-protein and the globular portion of the proteoglycan monomer were isolated from a trypsin-treated cartilage extract and were separated from each other by subsequent gel chromatography on Sepharose 6B and Sephacryl S-200. The affinity technique was also used for the preparation of these proteins labelled with dansyl groups.
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PMID:Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding properties from cartilage. 48 27

Media from cultured mouse peritoneal macrophages were tested for cartilage proteoglycan degrading activity using S35-labelled rabbit ear cartilage. Media samples collected at 2-day intervals contained increasing amounts of activity between days two and six. This activity was activated by trypsin and antagonized by chelating agents. The macrophage products induced release of the proteoglycan component of cartilage as determined by biochemical and histological methods without affecting the collagen component. Media from cells incubated with hydrocortisone were devoid of proteoglycan degrading activity.
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PMID:Release of cartilage proteoglycan degrading enzyme activity by thioglycollate stimulated mouse peritoneal macrophages in culture. 54 72

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.
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PMID:The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis. 60 25

1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 X 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 X 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.
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PMID:Endocytosis of sulphated proteoglycans by cultured skin fibroblasts. 74 44

Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the D1 preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average Mr = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average Mr = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% non-sulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45 degrees C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to penta- and hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfate-peptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.
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PMID:Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. 76


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