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)

In order to study the localization of Lentil lectin (LCH)-binding glycoresidues in glomeruli from patients with a variety of glomerulopathies, and to elucidate the relationship between LCH-binding sugars and the components of the extracellular matrix, laminin and type IV collagen, investigations of formalin-fixed, paraffin-embedded kidney tissues digested with trypsin were carried out by the direct and indirect immunofluorescence microscopy techniques. The glomerular basement membrane (GBM) and the mesangium reacted well with LCH, whereas areas with sclerotic lesions exhibited a decreased reactivity. The pattern of LCH binding to the GBM in various glomerulopathies was similar to that of laminin but different from that of type IV collagen. The pattern of localization of LCH-reacting sites and of laminin in the GBM included the double linear lines in diabetic nephropathy, inner linear line with outer projections (spikes) in membranous nephropathy, and reduplicated basement membrane in membranoproliferative glomerulonephritis. The results obtained by enzyme-linked immunoadsorbent assay showed that LCH had a stronger reactivity for laminin than for type IV collagen or fibronectin. These findings suggest that LCH is more reactive with laminin than with other components of the glomerular extracellular matrix.
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PMID:Histochemical and immunohistochemical studies of diseased human glomeruli. 203 28

The present study was designed to obtain further information on the nature of the corneal macromolecule(s) to which Pseudomonas aeruginosa adheres and how adherence might be prevented. Scarified adult mouse corneas in organ culture were treated with trypsin or lipase to determine whether the receptor molecule(s) was protein or lipid in nature. Trypsin (20 micrograms/ml) treatment of the cornea for 5 min had no significant effect on bacterial adherence, and longer periods of enzyme exposure resulted in extensive surface cell lysis. In contrast, lipase treatment (50,000 U/ml) for 1 h caused little visible cell lysis and significantly reduced bacterial adherence. To test further the lipid nature of the receptor, a highly purified monosialoganglioside (GM1) preparation (500 micrograms/ml) was used to preincubate (1 h) the cornea prior to bacterial application, and this also inhibited bacterial adherence. Similar corneal treatment with gangliotetraosylceramide (asialo GM1) (500 micrograms/ml) had little effect on ocular bacterial binding. Premixing of the bacterial inoculum with GM1 prior to corneal application had no significant effect on inhibiting bacterial binding, but similarly premixing the bacterial inoculum with asialo GM1 transiently decreased adherence. Lastly, premixing of the bacterial inoculum or preincubation of corneas with fibronectin (500 micrograms/ml for 1 h) both decreased bacterial adherence. These findings provide evidence that the receptor-adhesin interactions of P. aeruginosa at the ocular surface in organ culture are complex, involve a glycolipid moiety, and may be blocked by a ganglioside containing at least one sialosyl residue or by fibronectin, which may bind to membrane-associated gangliosides.
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PMID:Characterization of Pseudomonas aeruginosa adherence to mouse corneas in organ culture. 210 29

The structural stability of the hamster fibronectin receptor has been studied using limited proteolytic digestion and anti-(fibronectin receptor) monoclonal antibodies of known specificity. Treatment of the solubilized intact receptor or of the dissociated alpha and beta chains with any one of several proteases generated large protease-resistant fragments (92-110 kDa). Western blot analysis of tryptic digests using subunit-specific monoclonal antibodies revealed the large trypsin-generated fragment to be of beta-subunit origin. No products from the alpha subunit were detected. The protease-resistant fragment is lost upon exposure to reducing conditions; thus, the highly disulfide-bonded region of the beta subunit is important in the maintenance of the tertiary structure of the entire subunit. In contrast to solubilized fibronectin receptor, membrane-bound receptor is much more stable to proteolysis, and tryptic cleavage results in two large immunoreactive fragments of approximately 100 kDa and 95 kDa. This suggests a difference in the conformation and/or oligomeric organization of the membrane-bound receptor as compared with the solubilized heterodimeric receptor.
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PMID:Protease resistance of the beta subunit of the hamster fibronectin receptor. Evidence for differential cleavage of membrane-bound and soluble receptor. 213 10

The adherence of Treponema denticola GM-1, TD-4, and MS25 to human gingival fibroblasts (HGFs) was studied to serve as an introduction to investigations into the interactions of these oral bacteria with human host cells. Under both aerobic (5% CO2) and anaerobic (85% N2 plus 10% H2 plus 5% CO2) environments, the interactions with the HGFs were such that strains GM-1 and MS25 were consistently more adherent than strain TD-4. Polyclonal antibodies to GM-1 inhibited GM-1 adherence by 70%, while MS25 and TD-4 showed differing degrees of cross-reactive inhibition, indicative of common but not identical epitopes on the surface of the three T. denticola strains. Pretreatment of the three strains with trypsin did not inhibit adherence; proteinase K did, however, inhibit this interaction by 80%. Trypsin pretreatment of the HGFs resulted in increases in adherence of 50 and 86% for GM-1 and MS25, respectively, while a decrease of 41% was noted for TD-4. Exposure of the T. denticola strains to sugars and lectin pretreatment of the HGFs implicated adherence mediation by mannose and galactose residues on the HGF surface. Periodate treatment of HGFs resulted in a 50% drop in adherence for GM-1 and MS25, but did not decrease that of TD-4. Addition of fetal bovine serum inhibited adherence of the three strains to differing degrees, with TD-4 being the most susceptible. Addition of purified fibronectin (100 micrograms/ml) resulted in greater than 50% inhibition in GM-1 and MS25 adherence, while a 25% increase occurred with TD-4. While strain differences were noted in some of the parameters studied, the results indicate two possibilities for T. denticola-HGF adherence: a lectinlike adhesin(s) on the T. denticola surface with affinity for galactose and mannose on the HGF surface, and a serum host factor(s) bridging T. denticola and HGFs.
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PMID:Interaction of Treponema denticola TD-4, GM-1, and MS25 with human gingival fibroblasts. 216 Apr 30

Modulation of the synthesis and secretion of extracellular matrix proteins and matrix-degrading metalloproteases by rabbit synovial fibroblasts is an important model system for studying the control of tissue-specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin-treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin-treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well-formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron-microscopic views of detergent-extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide-treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin-treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with evidence that both induction and sustained expression of matrix-degrading metalloprotease may involve signals transduced through plasma membrane matrix receptors (integrins).
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PMID:Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase-inducing agents. 216 40

Fibronectin (FN), a glycoprotein present in the plasma and the extracellular matrix, has been shown to enhance adherence-related functions of polymorphonuclear leukocytes (PMNs). In this study we investigated the effects of FN on the activation of human PMNs in suspension by soluble stimuli, as determined by the generation of superoxide radicals (respiratory burst). FN (up to 100 micrograms/ml) did not directly stimulate the PMN respiratory burst assessed using a sensitive assay, luminol-dependent chemiluminescence (CL). Low FN concentrations (Up to 25 micrograms/ml) caused a dose-dependent enhancement of the CL induced by two chemoattractants. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet-activating factor (Paf), and also by phorbol myristate acetate (PMA), a known protein kinase C activator. Higher FN concentrations were less effective. The potentiation involved both initial rate and total CL responses and was more active on extracellular than intracellular generation of oxygen radicals. FN potentiation persisted after cell washing and was abolished by treatment of FN with trypsin. Measurement of the respiratory burst using the cytochrome c reduction assay confirmed that FN enhanced both the initial rate and total amount of superoxide anion generated by FMLP-stimulated PMNs. These data indicate that FN facilitates the respiratory burst of chemoattractant-stimulated PMNs and suggest that FN can prepare PMNs in suspension for amplified biological functions induced by soluble inflammatory stimuli.
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PMID:Priming effect of fibronectin on respiratory burst of human neutrophils induced by formyl peptides and platelet-activating factor. 217 7

To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin:collagenase dissociation in the absence of calcium. This aggregation is largely Ca2(+)-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2(+)-free trypsin:collagenase dissociation, aggregation of the surviving population is exclusively Ca2(+)-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K = 10(3) Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5 K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregation-inhibiting antiserum, and is itself capable of inhibiting CD limb bud cell aggregation. Fab fragments prepared from rabbit antisera specifically directed against the affinity-purified material also inhibit CD limb bud cell aggregation and this inhibition is neutralized by the 8.5 K protein. Our data thus demonstrate that CD limb bud cell aggregation is not mediated by fibronectin and/or collagen type I and indicate that this process is governed by a novel 8.5 K cell adhesion molecule.
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PMID:An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture. 222 2

Different procedures of enzymatic digestion of rat prostatic tissue and unique sets of mitogenic factors made it possible to culture practically pure populations of epithelial and stromal cells without previous separation of the two types of cells. Keratin-positive epithelial cells dissociated by trypsin and collagenase from adult rat ventral prostate proliferated in medium WAJC 404 supplemented with epidermal growth factor, insulin, cholera toxin, and bovine pituitary extract. Proliferation of epithelial cells was completely inhibited by dexamethasone as low as 30 nM. On the other hand, fibroblast-like stromal cells released by trypsin digestion required a plastic substratum coated with calf serum or fibronectin, and proliferated in Eagle's minimum essential medium supplemented with cholera toxin, bovine pituitary extract, dexamethasone, and bovine serum albumin. Epidermal growth factor and insulin had negligible effect on proliferation of stromal cells. Physiological concentrations of dihydrotestosterone and estradiol showed no effect on proliferation of both types of cells.
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PMID:Differences in growth requirements between epithelial and stromal cells derived from rat ventral prostate in serum-free primary culture. 223 29

A metalloproteinase with Mr 29,000 was purified to homogeneity as a latent proenzyme from the conditioned medium of a human rectal carcinoma cell line CaR-1. This enzyme hydrolyzed casein more potently than gelatin embedded in polyacrylamide gels in zymography assay. Calcium ion was essential for the activity. It exerted the maximum activity at pH 7-9. Its activity was stimulated by organomercurials, such as p-amino-phenyl mercuric acetate and p-chloromercuric benzoic acid, and was inhibited by 1,10-phenanthroline but was hardly affected by diisopropyl fluorophosphate and pepstatin. When the purified proenzyme was activated by the organomercurials, it effectively hydrolyzed fibronectin, laminin, type IV basement membrane collagen, and several types of gelatins but not interstitial type I and III collagens. The treatment of the purified proenzyme with p-aminophenyl mercuric acetate or trypsin formed an active peptide with Mr 20,000. The structural analysis indicated that it was most likely identical to putative metalloproteinase-1, the complementary DNA of which had been cloned from human tumor mRNAs capable of hybridizing to a rat transin complementary DNA. Based on the fact that this enzyme is secreted extracellularly and degrades the matrix proteins, we propose the name "matrin" for this newly identified enzyme.
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PMID:Purification and characterization of extracellular matrix-degrading metalloproteinase, matrin (pump-1), secreted from human rectal carcinoma cell line. 225 19

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96


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