Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Attachment of [35S]methionine-labelled mammalian type 3 reovirus to murine L cells and human HeLa cells was studied under equilibrium conditions. Cellular attachment sites could be completely saturated with 35S-labelled reovirus, indicating that specific attachment sites for reovirus are present on the surface of these cells. We calculated that L cells possess about 86000-105000 attachment sites per cell while HeLa cells possess about 126000-147000 sites per cell for type 3 reovirus. Unlabelled reovirus was highly efficient in competing for attachment by 35S-labelled reovirus to the saturable attachment sites of both L and HeLa cells, further indicating the specificity of the interaction. We also found that unlabelled reovirus competed equally well for both binding and internalization of 35S-labelled reovirus into murine L cells, suggesting that the L cell attachment site may serve as a virus entry site. Phospholipase digestion of L cells had no effect on subsequent reovirus attachment, while treatment of L cells with moderate concentrations of bromelain (but not trypsin, proteinase K or pronase) and Vibrio cholerae neuraminidase reproducibly decreased subsequent reovirus attachment. These results and those of others (Epstein et al., 1984, Virology 133, 46-55) suggest that mammalian reoviruses attach to specific cell surface receptors on at least two species of mammalian cells to initiate the infectious cycle.
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PMID:Saturable attachment sites for type 3 mammalian reovirus on murine L cells and human HeLa cells. 639 64

A procedure is described for purification of the primary bactericidal component of normal rabbit serum active in vitro against Bacillus subtilis. A 65 000-fold increase in specific bactericidal activity per milligram of serum protein was obtained, yielding a low molecular weight, heat-stable polypeptide fraction (PC-III) exhibiting biological activity at protein concentrations below 10 ng/mL. This preparation appeared homogeneous as judged by column chromatography and analytical NaDodSO4-polyacrylamide gel eletrophoresis; recovery of serum bactericidal activity was routinely greater than 80%. Analysis of dansylated or 125I-labeled samples in peptide-resolving polyacrylamide gels revealed a single band with an Mr of 1800. Optimal antibacterial activity of PC-III against B. subtilis occurred at an ionic strength of 0.24 and was absolutely dependent upon divalent cations; calcium was the most effective. Under optimum conditions, 4 ng/mL of PC-III reduced the viability of B. subtilis test innocula by 90% within 10 min at 37 degrees C. Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium were all sensitive to the action of PC-III, but higher bactericide concentrations were required to produce similar reductions in viability as observed with B. subtilis. All strains were killed by PC-III concentrations well below 1 microgram/mL, roughly that found in normal serum. The activity of PC-III preparations was significantly reduced by pretreatment with trypsin or proteinase K but not by neuraminidase or periodate.
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PMID:Antibacterial peptide from normal rat serum. 1. Isolation from whole serum, activity, and microbicidal spectrum. 679 8

Model systems simulating the cementum portion of teeth were used to characterize the attachment process by which certain species of oral Cytophaga initiate the colonization of the tooth root surface in vitro. The adsorption of these bacteria to spheroidal hydroxyapatite beads and mechanically powdered root material followed Langmuir isotherm kinetics. From such data, the number of binding sites per 20 mg of substrate and the affinity constants were evaluated for two strains of Cytophaga sp. Resting cells of the two strains tested adhered relatively tenaciously to hydroxyapatite beads in numbers similar to those observed with cells of Streptococcus sanguis. Attachment of bacteria to the substrates was partially inhibited by (i) coating the substrates with human serum or saliva, (ii) pretreating cell suspensions with proteinase K or phospholipase C or D, or (iii) exposing the cells to temperatures greater than 60 degrees C for 15 min. Treating resting cell suspensions with pronase, neuraminidase, phospholipase A2, or 0.1 M ethylenediaminetetraacetic acid had no effect on the attachment process.
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PMID:Attachment of oral Cytophaga species to hydroxyapatite-containing surfaces. 721 36

Binding of 125I-heparan sulphate was a common property of Helicobacter pylori strains isolated from patients with gastroduodenal ulcer diseases. Binding was (i) saturable; (ii) reversible by the addition of unlabelled heparan sulphate and heparin; (iii) inhibited by unlabelled heparan sulphate, heparin, and heparin oligosaccharides but not by other glycosaminoglycans of comparable size (chondroitin sulphate and dermatan sulphate) or by highly glycosylated glycoproteins (hog gastric mucin and fetuin); (iv) reduced by heat treatment (80 degrees C, 10 min) and exposure of the bacteria to pronase E, proteinase K, trypsin and chymotrypsin, but unaffected by treatment with pepsin and neuraminidase; and (v) time-, pH-, and ionic strength-dependent. Scatchard plot analysis of the binding data indicated the presence of one class of high-affinity receptor (Kd = 9 x 10(-9) M) for heparan sulphate.
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PMID:Affinity of the gastric pathogen Helicobacter pylori for the N-sulphated glycosaminoglycan heparan sulphate. 768 21

Interaction of Anaplasma marginale initial bodies with the bovine erythrocyte surface was examined by a direct hemagglutination assay. Purified initial bodies were shown to specifically hemagglutinate bovine erythrocytes but not erythrocytes from nonhost animal species. Hemagglutination was inhibited by treatment of purified initial bodies with trypsin, alpha-chymotrypsin, or proteinase K but not by treatment with neuraminidase or sodium periodate. Treatment of bovine erythrocytes with alpha-chymotrypsin or neuraminidase partially inhibited hemagglutination of the treated cells by initial bodies. In contrast, no inhibition occurred after treatment of erythrocytes with trypsin, phospholipases, or sodium periodate or when monosaccharides and disaccharides were used as potential competitive inhibitors. Thus, the initial body receptor is probably a surface protein, whereas the bovine receptor may comprise both protein and carbohydrate. Hemagglutination was unaffected by treatment of initial bodies with monoclonal or polyclonal antibodies raised against the A. marginale 31-kDa (MSP4) major surface polypeptide or non-A. marginale proteins or by treatment with a monoclonal antibody to the A. marginale MSP1a neutralization-sensitive epitope. In contrast, antiserum raised against whole A. marginale initial bodies or monospecific antibodies raised against purified A. marginale major surface polypeptides with molecular sizes of 105 (MSP1a), 100 (MSP1b), 61, and 36 (MSP2) kDa completely or partially inhibited hemagglutination. These data confirm the proposed surface location of the proteins susceptible to inhibition and suggest that they mediate hemagglutination of bovine erythrocytes. We propose that these surface proteins are possible adhesins.
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PMID:Characterization of hemagglutinating components on the Anaplasma marginale initial body surface and identification of possible adhesins. 792 25

Binding of canine parvovirus (CPV) to the susceptible feline T cell line 3201 was quantitated by fluorescence-activated cell sorter (FACS) analysis. CPV bound to the cells in a dose-dependent manner, while no binding to the non-permissive MSB-1 avian lymphoma cell line was detected. Binding could be competitively inhibited by addition of excess unlabeled empty capsids, or by pre-incubation of virus with a CPV-specific monoclonal antibody. To characterize the biochemical nature of this binding, live cells were treated with a variety of enzymes prior to use in the binding assay. Treatment with neuraminidase removed a significant proportion of the wild-type virus binding activity, while both proteinase K and phosphatidylinositol-specific phospholipase C (PI-PLC) prevented binding of a non-hemagglutinating (non-HA), non-sialic acid binding mutant to 3201 cells. This suggests that CPV binds to sialic acid expressed on host cells as well as erythrocyte membranes, and that it also binds a protein moiety which is glycosylphosphatidylinositol (GPI)-anchored. The role of these components in CPV infection was also examined by pretreating cells with neuraminidase or PI-PLC prior to inoculating them with either wild-type CPV or the non-hemagglutinating mutant. Neuraminidase treatment had no effect on the ability of CPV to infect the cells, while infectivity was severely compromised by pretreating the cells with either proteinase K or PI-PLC. GPI-anchored proteins on 3201 cells were further characterized by Triton X-114 extraction and reactivity to anti-CRD after PI-PLC treatment.
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PMID:Characterization of canine parvovirus (CPV) interactions with 3201 T cells: involvement of GPI-anchored protein(s) in binding and infection. 808 Dec 56

The distribution of anionic microdomains has been described in cerebral vessels and more recently in capillaries of peripheral nerve. Evidence is accumulating that these sites play a role in the barrier function of vascular endothelia in the PNS and CNS. The chemical nature of anionic sites has been at least partly determined for cerebral vessels but not in peripheral nerve. This study reports our preliminary investigations to determine the nature of endothelial anionic sites in sciatic nerve. The effects of digestion of ultra-thin sections of nerve with a battery of proteolytic and glycolytic enzymes (papain, trypsin, proteinase K, hyaluronidase, heparinase, heparitinase and neuraminidase) on the distribution of anionic sites was determined using the label, cationic colloidal gold. Papain, a proteolytic enzyme of broad specificity, succeeded in removing the majority of cationic colloidal gold-binding sites on the luminal surface of vascular endothelia. In contrast trypsin and proteinase K were less effective, reflecting their narrower specificity. Hyaluronidase, heparinase and heparitinase did not significantly affect cationic colloidal gold-labelling. However, a considerable reduction in cationic colloidal gold-binding occurred following neuraminidase digestion. These results suggest that, as in cerebral vessels, sialic acid-containing glycoproteins are largely responsible for the negatively charged domains on the luminal membrane of endothelial cells in peripheral nerve.
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PMID:Molecular characterization of anionic sites on the luminal front of endoneurial capillaries in sciatic nerve. 817 16

Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria. Furthermore, human colostrum secretory IgA inhibited adhesion in a metaperiodate- and alpha-L-fucosidase-sensitive but neuraminidase-independent fashion. The in situ adherence assay should be useful in further characterizing the H. pylori adhesin and its receptor and for identifying therapeutically useful compounds that inhibit strain-specific and cell lineage-specific binding of this human pathogen.
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PMID:An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium. 838 33

Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.
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PMID:Tenascin supports lymphocyte rolling. 915 79

Among the wall-less mycoplasmas only a few species have been identified with a capsule at their cell surface. Mycoplasma penetrans is a recently identified mycoplasma with unique morphology, isolated from HIV-infected patients. Using transmission electron microscopy, it was found that M. penetrans is surrounded by capsular material 11 nm (strain GTU-54-6A1) to 30 nm (strain HF-2) thick, which can be stained with ruthenium red and labelled with cationized ferritin. The polysaccharide composition of this capsule was indicated by its staining with periodic acid-thiocarbohydrazide silver proteinate and the abolition of ruthenium red staining of the cell surface by neuraminidase treatment. In addition, proteinase K treatment of the M. penetrans cells resulted in removal of the capsule, suggesting that polypeptides may contribute in anchoring it to the membrane or in its stability. Two different types of glycosylated material were detected in mycoplasma extracts by SDS-PAGE and periodic acid-Schiff staining. The first component was a high-molecular-mass material, which was heat- and proteinase-K-labile and which probably constitutes the capsular polymer. The other component was a low-molecular-mass glycolipid fraction, which was proteinase-K-, heat- and EDTA-resistant. The identification of a capsule at the M. penetrans cell surface is of particular interest for a mycoplasma which has been shown to adhere to various host cells and to penetrate into their intracellular compartments. The capsule may have significance in the pathogenesis of disease associated with infection by this organism.
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PMID:Identification of two glycosylated components of Mycoplasma penetrans: a surface-exposed capsular polysaccharide and a glycolipid fraction. 961 99


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