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)

The location, with respect to the membrane, of Lys 165 in the folded beta polypeptide of native nicotinic acetylcholine receptor has been determined by site-directed immunochemistry. Sealed, right-side-out vesicles rich in acetylcholine receptor were modified with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to one portion of the vesicles to make them permeable to the pyridoxal phosphate and sodium borohydride; the other portion was modified in the absence of saponin. Both samples were then exhaustively succinylated and digested with trypsin and thermolysin to produce the peptide LDAKGER, which contains Lys beta 165. The digests were passed over an immunoadsorbent specific for peptides with the sequence LDAXGER, where X represents any modified or unmodified amino acid, and specifically bound peptides were eluted with 0.1 M sodium phosphate, pH 2.5. The eluates were submitted to high-pressure liquid chromatography, and two peptides, N epsilon-phospho[3H]pyridoxalLDAKGER and N epsilon-succinylLDAKGER, modified at the epsilon amino group of lysine with pyridoxal phosphate and sodium [3H]-borohydride or succinic anhydride, respectively, were identified by comparison to standards. The relative specific radioactivity of N epsilon-phospho[3H]pyridoxalLDAKGER modified in the presence or absence of saponin, respectively, was 0.9 +/- 0.4. The incorporation of phospho[3H]pyridoxyl groups into Lys alpha 380, a residue located on the cytoplasmic surface of acetylcholine receptor, was also monitored. The relative specific radioactivity of the peptide that contains the modified Lys alpha 380, N epsilon-phospho[3H]pyridoxalGVKYIAE, increased 3.6-fold when the modification was performed in the presence of saponin. This result verifies that the vesicles used in these experiments were sealed and right-side-out. Because the incorporation of [3H]pyridoxyl groups into Lys beta 165 is the same in the presence or absence of saponin, Lys beta 165 must have been located on the outside surface of the sealed, right-side-out vesicles, and therefore on the extracytoplasmic surface of native acetylcholine receptor.
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PMID:Extracytoplasmic disposition of lysine beta 165 of acetylcholine receptor. 817 83

Agrin is a component of the synaptic basal lamina that induces the aggregation of acetylcholine receptors (AChRs) and other elements of the postsynaptic membrane. We have determined the localization, binding characteristics, and biochemical profile of the agrin receptor in Torpedo electric organ membranes and defined domains of agrin that bind this receptor. Postsynaptic membranes from Torpedo electric organ bind agrin as judged by depletion of AChR clustering activity from solution. A ligand-based radioimmunoassay shows that agrin binding to postsynaptic membranes is saturable and calcium-dependent. Half-maximal binding is observed at agrin concentrations < or = 10(-10) M. Identification of the bound agrin polypeptides shows that at least one membrane binding domain of agrin is located in a 70-kDa proteolytic fragment. Immunofluorescent visualization and radioimmunoassay of agrin binding demonstrates that the agrin receptor is selectively concentrated in postsynaptic membranes, with little binding detected on nonsynaptic or liver membranes. Agrin binding is greatly reduced if the membranes are pretreated with trypsin, but is unaffected by phosphatidylinositol-specific phospholipase C. Membranes stripped of peripheral proteins by alkaline treatment retain full ligand binding capacity. alpha-Bungarotoxin affinity columns bind AChRs but not agrin receptors. The ratio of agrin receptors to AChRs in postsynaptic membranes is approximately 1:200. We conclude that the agrin receptor is an integral membrane glycoprotein that is selectively concentrated in postsynaptic membranes, but that is not tightly complexed with the AChR. The results also indicate that the biological activity of agrin is mediated through intracellular signal transduction events triggered by ligand binding to the agrin receptor.
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PMID:The agrin receptor. Localization in the postsynaptic membrane, interaction with agrin, and relationship to the acetylcholine receptor. 822 74

Several postsynaptic neurotoxins (alpha-neurotoxins) with distinct pharmacological and biochemical properties were isolated and purified from the King cobra venom (Ophiophagus hannah) by employing sequentially preparative-scale cation-exchange chromatography on SP-Sephadex C-25 coupled with gel filtration and reversed-phase HPLC. The complete sequence of one neurotoxin was determined by N-terminal Edman degradation with the automatic pulsed-liquid phase sequencer on some peptide fragments generated from the endopeptidases, i.e. trypsin, S. aureus V8 protease and lysyl endopeptidase. This novel neurotoxin is a basic polypeptide of pI 9.05, consisting of 72 amino-acid residues with 10 cysteine residues. It is found to share about 60% sequence homology with Toxins a and b isolated from the same venom and the well established alpha-bungarotoxin, a major postsynaptic toxic ligand for acetylcholine receptor isolated from Bungarus multicinctus. The characterized alpha-neurotoxin molecules were also shown to bind specifically with nicotinic acetylcholine receptors of the electric eel, Torpedo californica.
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PMID:Sequence characterization of a novel alpha-neurotoxin from the king cobra (Ophiophagus hannah) venom. 844 23

The binding of rabies virus to cellular membranes was measured using an enzyme-linked immunosorbent assay (ELISA). Virus binding to membranes adsorbed to the wells of microtiter plates was detected with rabies virus antibody and alkaline phosphatase-linked second antibody. The greatest degree of binding was to myotube, neuroblastoma, and salivary gland membranes; intermediate levels occurred in striated muscle and nerve membranes; and low levels of binding were found in other membranes, including those of most parenchymal organs. Binding of rabies virus to myotube membranes was saturable, dependent on pH (with an optimum of pH 6.0), facilitated by the divalent cations Ca++, Mn++, and Mg++, and was temperature dependent. Binding was greatly reduced by inactivation of virus with beta-propiolactone or treatment of virus with trypsin. In embryonic chick myotubes, total acetylcholine receptor content and acetylcholinesterase activity undergo marked changes during development, first increasing and then decreasing at the time of hatching. Binding of rabies virus followed a similar pattern, indicating that the virus may interact with the acetylcholine receptor or other surface molecules undergoing similar developmental changes.
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PMID:Rabies virus binding to cellular membranes measured by enzyme immunoassay. 1675 1


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