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)

This study examined binding of Pseudomonas aeruginosa to unwounded postnatal day (P) 5 immature mouse cornea. To determine whether the receptor molecule was protein or lipid in nature, the eyes were incubated in vitro with trypsin or lipase before bacteria were applied. Trypsin significantly increased bacterial binding, whereas lipase had no effect. To determine if enhanced binding after trypsin indicated exposure of a lipid or a protein receptor, the eyes were treated sequentially with trypsin, then lipase. Subsequent binding did not differ significantly from trypsin alone, indicating that the exposed receptor was not a lipid. Incubation of the P 5 eye with neuraminidase to remove sialylated residues also significantly enhanced binding at all times, and premixing of the inoculum with the enzyme before adherence testing increased binding at 15 and 30 min. The effects of a monosialoganglioside (GM1) and gangliotetraosylceramide (asialo GM1) were also examined. Incubation of eyes with GM1 or asialo GM1 produced no significant inhibition of bacterial binding, but premixing of the bacterial inoculum with GM1 or asialo GM1 before corneal application transiently decreased adherence. Fibronectin (FN) treatment of the P 5 eye, premixing FN with the bacterial inoculum before its ocular application, or similar treatment with N-acetylneuraminic acid (NANA) transiently inhibited binding. These data demonstrate that pseudomonal binding to the unwounded eye is not lipase sensitive and is enhanced by trypsin treatment which exposes a lipase insensitive receptor and by neuraminidase which removes sialylated residues. It is not inhibited by pretreatment of the eye with either GM1 or asialo GM1 and is transiently inhibited by pretreatment of the eye or the inoculum with NANA or FN.
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PMID:Characterization of pseudomonal adherence to unwounded cornea. 190 77

Tritium-labeled neoglycolipids consisting of the oligosaccharide of ganglioside GM1 attached to cholesterol (GM1OSNH-X-CHOL), phosphatidylethanolamine (GM1OS-PE) and stearylamine (GM1OSNHC18) were synthesized and their uptake and metabolism by GM1-deficient rat glioma C6 cells were determined. When the neoglycolipids were added to serum-free culture medium, all three were rapidly taken up by the cells and initially inserted into the plasma membrane based on their resistance to trypsin and their ability to bind cholera toxin. With time, the neoglycolipids underwent internalization as the ratio of cell-associated radioactivity to cell surface toxin binding increased; this process was slow for GM1OSNH-X-CHOL and GM1OS-PE and rapid for GM1OSNHC18. Analysis of lipids extracted from the cells indicated that the neoglycolipids also underwent metabolism to GD1aOS-based analogues. In addition, GM1OSNH-X-CHOL and GM1OSNHC18 were degraded to their GM2OS-based analogues, whereas GM2OS-PE was not detected. In contrast, large amounts of 3H were recovered in the medium from cells treated with GM1OS-PE and the label was associated with material that behaved neither as an oligosaccharide or a neoglycolipid. In the presence of monensin or chloroquine, metabolism of the three neoglycolipids was inhibited. Thus, GM1OS-based neoglycolipids were taken up by the cells, internalized and sorted both to the Golgi apparatus (sialylated to GD1aOS-based analogues) and to lysosomes (hydrolyzed to GM2OS-based analogues). The rate and extent of these processes, however, were strongly influenced by the nature of lipid moiety.
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PMID:Metabolism of cholesterol, phosphatidylethanolamine and stearylamine analogues of GM1 ganglioside by rat glioma C6 cells. 203 49

Botulinum A neurotoxin (BoNtx) produced a partial inhibition of carbachol induced 3H-noradrenaline (3H-NA) release from bovine adrenal chromaffin cells in monolayer culture. Each of the polysialogangliosides GD1a, GT1b and GD1b enhanced the block of exocytosis when they were applied prior to the toxin exposure. The monosialoganglioside GM1 was not effective. Chromaffin cells treated with neuraminidase lost their sensitivity to BoNtx. Application of gangliosides to these cells, however, restored their susceptibility to the toxin. Treatment of the cells with trypsin did not affect the BoNtx-blockade of 3H-NA-release. The potency of botulinum A toxin was increased in a solution of low ionic strength in which sodium chloride was replaced by sucrose. In agreement with the potency of botulinum A neurotoxin in blocking exocytosis under the various conditions, binding of 125I-botulinum A neurotoxin to chromaffin cells was enhanced in low ionic strength solution and by pretreatment of the cells with gangliosides. The binding was decreased by digestion of gangliosides with neuraminidase. It is concluded that botulinum A neurotoxin binds exclusively to polysialogangliosides which subsequently serve as carriers for the toxin. The low ionic strength may increase some physico-chemical interaction of the toxin with the polysialogangliosides.
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PMID:The translocation of botulinum A neurotoxin by chromaffin cells is promoted in low ionic strength solution and is insensitive to trypsin. 204 36

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

Cultured HeLa cells were incubated with pyrene-GM1/3H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of 3H-GM1. The pyrene-GM1 and 3H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and 3H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GD1a) derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca2+ salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca2+ concentration dependent (maximum at 10 mM), and subsequent addition of EDTA had no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties.
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PMID:Association to HeLa cells and surface behavior of exogenous gangliosides studied with a fluorescent derivative of GM1. 211 Aug 27

GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.
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PMID:Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin. 215 9

A 68,000-molecular-weight protein was isolated by polyacrylamide gel electrophoresis from the organism-free filtrate of a fully virulent clinical strain of Campylobacter jejuni. The eluted protein was heat labile, was inactivated at either pH 3.0 or 9.0, was sensitive to trypsin, and was lethal for fertile chicken eggs. It also had toxic effects on chicken embryo fibroblast, Chinese hamster ovary (CHO), and intestinal 407 (Int407) cells. A monoclonal antibody (CETPMAb4) raised to this eluted toxic protein (ETP) from C. jejuni abolished these toxic activities. Homology between C. jejuni ETP and Vibrio cholerae toxin was not observed in that specific antisera to each did not block their respective toxic activities. In enzyme-linked immunosorbent assays, ETP, unlike chlorea enterotoxin, did not bind to GM1 ganglioside. Furthermore, the C. jejuni toxin had cytotoxinlike properties and induced rounding of CHO cells. Binding of ETP to Int407 and primary chicken embryo fibroblast cells was maximal after 2 h as assessed by enzyme-linked immunosorbent assay, and this toxin adherence to host cell membranes was significantly reduced by prior treatment of the cells with proteolytic enzymes, neuraminidase, or glutaraldehyde but not by treatment with beta-galactosidase, lipase, Nonidet P-40, or sodium metaperiodate. In competitive binding assays, sugars, lectins, or GM1 ganglioside did not adversely influence uptake of ETP by these cells. These results suggest that the ETP produced by C. jejuni is a cytotoxin which binds to Int407 cells via a protein- or glycoproteinlike receptor on cell membranes and possesses properties dissimilar to those of V. cholerae toxin.
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PMID:Isolation, characterization, and host-cell-binding properties of a cytotoxin from Campylobacter jejuni. 219 97

Binding of type C neurotoxin (C1 toxin) from Clostridium botulinum (strain Stockholm) to neuroblastoma cell lines was studied by using biotinylated anti-toxin antibody and avidin-biotinylated peroxidase complex. The neurotoxin bound with high efficiency to mouse neuroblastoma (NS-20Y and NIE-115) cells and to hybridomas of rat glioblastoma and mouse neuroblastoma (NG108-C15) cells. The toxin bound little to human neuroblastoma, rat astrocytoma, and nonneural cell lines. Binding of the neurotoxin to NG108-C15 cells was inhibited by gangliosides (GT1b and GM1) and by monoclonal antibodies (CA-12 and C-9), although inhibition was not complete. Sequential preincubation of C1 toxin with GT1b and CA-12 caused complete inhibition. A Scatchard plot of binding of 125I-labeled C1 toxin to NG108-C15 cells showed a hyperbolic curve. Monoclonal antibody CA-12 but not C-9 neutralized the lethal activity of the toxin toward mice. Only C-9 clearly inhibited toxin binding to GT1b. These results suggest that NG108-C15 cells have at least two kinds of receptors for C1 toxin. From the results of binding tests with neuraminidase-, pronase-, and trypsin-treated NG108-C15 cells, the chemical nature of the high-affinity site was presumed to be a glycoprotein containing sialic acid. GT1b may have an important role in low-affinity sites.
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PMID:Binding of Clostridium botulinum type C neurotoxin to different neuroblastoma cell lines. 253 34

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylgluc osyl ceramide (GM1) on high-affinity binding of L-[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40-50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H-labeled GM1 proved trypsin-stable insertion of GM1 into SPM, with a maximum incorporation of four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+-stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 microliters/mg of protein, in ganglioside-treated as well as in control membranes. From the data, it is concluded that a special, Na+- and Cl- -independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.
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PMID:Glutamate transport and not glutamate receptor binding is stimulated by gangliosides in a Ca2+-dependent manner in rat brain synaptic plasma membranes. 256 1

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10(-4) M [3H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca2+ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [3H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.
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PMID:Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: inhibition by calcium ion and dependence upon membrane protein. 266 79


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