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)

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A soluble component of about 50 kDa from embryonic eye tissue, the synaptic target of the ganglion, increases the development of ACh sensitivity by the neurons 10-fold over a 1-week period in culture. The increased sensitivity does not arise from a change in agonist affinity or esterase activity. Both the basal ACh response obtained in the absence of the 50-kDa component and the elevated responses obtained with it can be inhibited by neuronal bungarotoxin (nBgt) but not by alpha-bungarotoxin (alpha Bgt). Increases of less than twofold are observed for the binding of anti-AChR monoclonal antibody 35 (mAb 35), nBgt, and alpha Bgt to the neurons under these conditions. Extract fractions containing the 50-kDa component also enable the neurons to enhance their ACh responses through a cAMP-dependent mechanism. Either the 50-kDa fraction induces the appearance of a new type of AChR regulated by cAMP, or it alters the function of existing AChRs. The 50-kDa fraction produces no change in neuronal growth but can increase GABA responses sixfold, indicating that its effects are not confined to AChRs. It is not clear whether a single molecular species is responsible for the diverse regulatory effects or whether several types of active components are present in the fraction. The component which enhances ACh sensitivity is trypsin-sensitive and heat-labile, as expected for a protein. The component may be widely distributed since the 50-kDa fraction from a number of tissues can increase the ACh response. The fraction from eye tissue, however, has a specific activity 5-10 times greater than that of the liver fraction. A wide distribution would suggest multiple targets and roles for the component during development.
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PMID:Regulation of acetylcholine receptors on chick ciliary ganglion neurons by components from the synaptic target tissue. 164 5

To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.
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PMID:Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite. 174 30

The native, membrane-bound, acetylcholine receptor from Torpedo marmorata was photolabeled by the competitive antagonist p-[3H]dimethylaminobenzene-diazonium fluoroborate (DDF) in the presence of the noncompetitive blocker phencyclidine and under energy transfer conditions. The isolated alpha-subunits were treated with cyanogen bromide and fractionation of the resulting fragments yielded three radiolabeled peptides, at the level of which, incorporation of [3H]DDF (i) was equally inhibited by the agonist carbamoylcholine and the competitive antagonist alpha-bungarotoxin and (ii) was insensitive to "scavenging" reagents. Subfragmentation of cyanogen bromide peptide III with omicron-iodosobenzoic acid or trypsin and sequence analysis of the fragments led to the identification of a novel amino acid alpha-Tyr-93 (and possibly Trp-86) as labeled by [3H]DDF in a carbamoylcholine-sensitive manner. alpha-Tyr-93 is conserved in the muscle and neuronal alpha-subunits but not in the other subunits of muscle receptor. This result provides evidence for a site involving at least a third loop of the alpha-subunit amino-terminal hydrophilic domain, in addition to the ones previously identified (Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, J. Y., Lazure, C., Chretien, M., and Changeux, J. P. (1988) Biochemistry 27, 2346-2357). Possible contribution of tyrosine side-chains to the complexation of the quaternary ammonium group of cholinergic ligands is discussed.
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PMID:Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites. 235 8

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker ]3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity side for noncompetitive blockers. The alpha-subunit was purified and digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the alpha-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta-chain [1] and Ser-254 and Leu-257 in the beta-chain [2]. Extended sequence analysis of the hydrophobic segment M1 further showed that no labeling-occurred in this region.
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PMID:The noncompetitive blocker [(3)H]chlorpromazine labels segment M2 but not segment M1 of the nicotinic acetylcholine receptor alpha-subunit. 247 58

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.
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PMID:Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor. 274 50

By a mild and highly reproducible fractionation of Torpedo californica electric tissue, we prepared membrane which was 30 times enriched in nicotinic acetylcholine receptor (AChR). This preparation was neither alkali-stripped nor reconstituted and consequently contained nu (43-kDa protein), which is associated with the cytoplasmic aspect of the receptor. We tested this membrane for the presence of sealed vesicles and determined the orientation of these vesicles by combining three methods. Two of these methods were based on the accessibilities, in the presence and absence of detergent, of the extracellular acetylcholine binding site to alpha-bungarotoxin and of the intracellular nu to trypsin. These two methods are specific for AChR-containing membrane. The third method was morphometry of electron micrographs, by which we estimated the proportion of sequestered membrane. These methods taken together indicated that approximately 45% of the AChR-containing membrane was in the form of leaky vesicles or sheets, 33% was sealed right-side-out vesicles, 11% was sealed inside-out vesicles, and 11% was sequestered within multilamellar or multivesicular vesicles. The complexity of this membrane needs to be taken into account in sidedness studies of the AChR.
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PMID:The intactness and orientation of acetylcholine receptor-rich membrane from Torpedo californica electric tissue. 275 9

Chemical modification of the Torpedo californica acetylcholine receptor (AcChR) by the fluorescent agent N-(1-pyrenyl)maleimide (PM) under nonreducing conditions resulted in the labeling of cysteine residues in all subunits and marked inhibition of the AcChR ion channel opening [Clarke, J. H., & Martinez-Carrion, M. (1986) J. Biol. Chem. 261, 10063-10072]. The PM alkylation kinetics are not affected by the presence of agonists or a competitive antagonist. The PM-labeled alpha-subunit has been purified and digested with both CNBr and trypsin. The resulting fragments from both cleavages were fractionated by high-performance liquid chromatography. The amino acid analysis and sequencing data of the PM-labeled peptides identified cysteine-222 as the only residue labeled by PM on the alpha-subunit primary structure. Cysteine-222 is located in the middle of a hydrophobic domain designated M1, which contains a homologous class of cysteines (Cys-241 in the aligned sequences) conserved in the four subunits of the AcChR. Because of its reactivity and fluorescent properties of the bound probe, alpha Cys-222 seems to be free sulfhydryl group accessible through a hydrophobic pocket, and these properties should be incorporated into proposed folding models for the alpha-subunit.
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PMID:Covalent modification of a critical sulfhydryl group in the acetylcholine receptor: cysteine-222 of the alpha-subunit. 281 78

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The delta subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and cosequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [3H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII.
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PMID:Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine. 308 4

Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.
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PMID:Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. 345 58

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled beta chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.
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PMID:Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the beta and delta chains. 360 23


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