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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nicotinic acetylcholine receptor (AcChR) was purified from fetal calf muscle by an affinity chromatographic method utilizing alpha-neurotoxin from Naja naja siamensis as an immobilized ligand. Preparations of AcChR with an average specific activity of 5 nmol of alpha-toxin bound/mg of protein were obtained, i.e., 75% of the theoretical specific activity assuming identity with Torpedo AcChR. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified AcChR consistently showed the presence of five polypeptides, having apparent Mr's of 42 000, 44 000, 49 000, 55 000, and 58 000, respectively. The peptide of Mr 44K was demonstrated to be actin. The amino acid composition of fetal calf AcChR was shown to be similar to that of Torpedo AcChR. In addition, calf AcChR contained large amounts of amino sugars. The sedimentation coefficient of the purified calf AcChR was found to be 9.25 +/- 0.25, i.e., similar to the monomeric form of electric organ AcChR. Determination of the isoelectric point of alpha-bungarotoxin/calf AcChR complexes revealed the presence of two charged forms, having pI values of 5.16 +/- 0.13 and 6.05 +/- 0.18, respectively.
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PMID:Mammalian muscle acetylcholine receptor purification and characterization. 710 16

Acetylcholine receptors were purified to homogeneity from chicken embryonic, adult innervated and denervated muscles, by bio-specific chromatographies using immobilised alpha-neurotoxin and lentil lectin. A minimum specific activity for the pure receptor was estimated to be 6000 nmol alpha-toxin binding sites/g protein. For analysis, the receptors were radio-iodinated or tritiated to high specific radioactivity with succinimidyl-[2,3-3H]propionate. All of the iodinated protein present in the purified receptor preparation reacted with antibody against the pure acetylcholine receptor from Torpedo marmorata electric organ. In the case of all three muscle types used the same oligomeric forms were obtained. The principal form has a sedimentation coefficient of about 9 S, while a minor species (approximately 5S) was also appreciable in crude preparations of embryonic and denervated muscles. Immunization of rabbits with the homogenous receptor from chicken denervated muscle produced muscle weakness characteristic of experimental autoimmune myasthenia gravis. These antisera were equally reactive towards the receptor --125I-alpha-bungarotoxin complexes from chick innervated and denervated muscles. Likewise, the electrophoretic mobilities of the receptors (9-S form) from all three muscle types were identical, as were the isoelectric points of their complexes with 125I-alpha-bungarotoxin. Collectively, these findings and associated ones on subunit structure denote that the 9-S receptor molecules from junctional and extra-junctional area and embryonic stage of chicken muscle are indistinguishable by all criteria yet applied to them.
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PMID:Similarity of acetylcholine receptors of denervated, innervated and embryonic chicken muscles. 1. Molecular species and their purification. 714 Jul 38

Nicotinic acetylcholine receptor protein has been purified from human skeletal muscle by a procedure involving extraction in non-ionic detergent followed by affinity purification on immobilised alpha-toxin. Purified receptor preparations had specific activities of 0.5-3.5 mumol alpha-bungarotoxin binding sites/g protein and sedimented as a single 125I-alpha-bungarotoxin-binding species in sucrose-density-gradient centrifugation with s20,w = 9.5 S. The purified protein focussed as a single sharp band at pH 5.1 when complexed to 125I-alpha-bungarotoxin. Polyacrylamide gel electrophoresis of the purified receptor under denaturing conditions showed two major protein bands with Mr 42 000 and 66 000 respectively with the occasional appearance of minor components of Mr 56 000 and 85 000. Only the 42 000-Mr band was labelled with the affinity reagent, 4-(N-maleimido)[3H]-benzyltrimethylammonium. The purified receptor bound 125I-alpha-bungarotoxin and d-tubocurarine with Kd values of 0.5 nM and 0.25 microM respectively. It behaved similarly to unpurified detergent-extracted human receptor in the radioimmunoassay for anti-(human acetylcholine receptor) antibodies and when injected into rabbits caused increased levels of the latter antibodies but did not cause experimental autoimmune myasthenia gravis.
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PMID:The isolation and characterisation of the nicotinic acetylcholine receptor from human skeletal muscle. 722 75

Multiple forms of the acetylcholine receptor solubilised from cat denervated muscle were separated by velocity sedimentation centrifugation. The kinetic properties of the two main forms (with sedimentation coefficients of 9 S and 4 S) were investigated using a pure preparation of a suitable probe, [3H]propionyl-alpha-bungaro-toxin. The binding of this toxin to each of these forms of the muscle receptor was consistent with a simple bimolecular reaction with a homogeneous class of binding sites. Negligible dissociation of the receptor-toxin complex was observed. This behaviour was also found for the different forms of the acetylcholine receptor of chick embryo muscle and of Torpedo marmorata electric organ. Association rate constants for binding of the 3H-labelled alpha-toxin to receptor from chick embryo muscle and the 9-S and 4-S forms from cat denervated muscle were 0.54 X 10(5), 1.76 X 10(5) and 2.69 X 10(5) M--1 S--1 respectively, at 25 degrees C. The values obtained for the 9-S and 13-S forms of receptor from T. marmorata were 4.51 X 10(5) and 9.93 X 10(5) M--1 S--1 respectively. The reaction of the 3H-labelled alpha-toxin with the receptor was second-order and linear in the presence of an antagonist, as in its absence, for the 4-S and 9-S forms of the cat denervated muscle receptor. This reaction of the receptor was inhibited by cholinergic ligands, with Ki values for two antagonists tested being greater with the 4-S form than with the 9-S form. Apparent negative interaction is observed for antagonists with this receptor, with Hill coefficients of about 0.78 and 0.64 for the 4-S and 9-S forms respectively. A ligand-induced affinity increase, produced by the agonists but not by the antagonists, was observed in this reaction for both forms of the muscle receptor. Two agonists tested showed no difference between these forms in their high-affinity states in either their binding affinities or Hill coefficients.
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PMID:Molecular forms of the acetylcholine receptor from vertebrate muscles and Torpedo electric organ. Interactions with specific ligands. 725 Jan 20

The alpha subunits of Gq family G proteins, GL1 alpha (G14 alpha), GL2 alpha(G11 alpha), and Gq alpha were expressed with G protein beta 1 and gamma 2 subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1 (GL1 alpha beta gamma), GL2 (GL2 alpha beta gamma), and Gq (Gq alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gq activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to GL1, GL2, and Gq in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of Gq family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 microM GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of GL1, GL2, and Gq in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of GO. Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.
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PMID:Characterization of Gq family G proteins GL1 alpha (G14 alpha), GL2 alpha (G11 alpha), and Gq alpha expressed in the baculovirus-insect cell system. 789 Jul 62

Chinese hamster ovary cells were transfected with both A1 adenosine receptor and muscarinic type 3 acetylcholine receptor cDNAs. The muscarinic receptor agonist carbachol stimulated phospholipase C activity, resulting in Ca2+ mobilization and arachidonate release. N6-Cyclopentyladenosine (CPA), an A1 receptor agonist, did not activate Ca(2+)-related signal transduction systems by itself but instead inhibited cAMP accumulation. In the presence of carbachol, however, the A1 receptor agonist enhanced muscarinic receptor agonist-induced phospholipase C/Ca2+ responses. In addition, the arachidonate release caused by Ca2+ ionophores or thapsigargin was also amplified by CPA, without a change in phospholipase C activity. Thus, CPA augments Ca(2+)-mediated phospholipase A2 activation in addition to and separate from its ability to amplify phospholipase C-mediated Ca2+ mobilization. Because the permissive actions of CPA on phospholipase C and phospholipase A2 activation were each reversed by pertussis toxin treatment, in a manner similar to that of the CPA-induced inhibition of cAMP accumulation, we conclude that a single species of A1 receptor expressed in Chinese hamster ovary cells can couple to multiple signal transduction systems stemming from phospholipase C stimulation, phospholipase A2-mediated and Ca(2+)-dependent arachidonate release, and inhibition of cAMP accumulation. A pertussis toxin-sensitive G protein (or proteins) mediates the permissive actions of the A1 receptor in the stimulation of phospholipase C- and phospholipase A2-mediated arachidonate release.
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PMID:A single species of A1 adenosine receptor expressed in Chinese hamster ovary cells not only inhibits cAMP accumulation but also stimulates phospholipase C and arachidonate release. 819 94

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

The sequence region 55-74 of the alpha-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment--residues alpha 67-76--forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence alpha 55-74 of Torpedo AChR binds alpha-bungarotoxin (alpha BTX), suggesting that amino acid residues within this sequence region may contribute to formation of an alpha BTX binding site. Using single-residue substituted synthetic analogues of the sequence alpha 55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with alpha-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg55, Arg57, Trp60, Arg64, Leu65, Arg66, Trp67, or Asn68 strongly inhibited alpha-toxin binding, whereas substitutions of Ile61, Val63, Pro69, Ala70, Asp71, or Tyr72 had marginal effects. Substitutions within the region alpha 68-72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp60 substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp62 slightly but consistently affected binding of mAbs 6 and 22.
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PMID:Amino acid residues within the sequence region alpha 55-74 of Torpedo nicotinic acetylcholine receptor interacting with antibodies to the main immunogenic region and with snake alpha-neurotoxins. 851 74

Muscarinic acetylcholine receptor (mAChR) activation in isolated cells from the nasal salt gland of the domestic duck (Anas platyrhynchos) results in a rapid increase in the rate of phosphatidylinositol hydrolysis and pronounced intracellular calcium signals. Both responses can be elicited by treating these cells with fluoroaluminate (AlF4-) indicating the involvement of a heterotrimeric G protein in the transmembrane signaling process. To characterize this G protein, electrophoretically separated membrane proteins were blotted onto nitrocellulose filters and probed with peptide-antibodies raised against portions of different alpha-subunits of mammalian G proteins. We could demonstrate the presence of at least four different G proteins in salt gland cell membranes. Two of these proteins (40 and 41 kD) were ADP-ribosylated by pertussis toxin and were recognized by an antiserum against a common sequence in all G protein alpha-subunits. One protein (46 kD) was a cholera toxin-substrate and was recognized by a Gs-specific antiserum; the other (42 kD) was recognized by Gq-specific antisera and was resistant to ADP-ribosylation. Since the initial inositol phosphate production upon receptor activation with carbachol and the resulting calcium signals were not affected by pertussis toxin-pretreatment of salt gland cells, we conclude that muscarinic receptors are coupled to phospholipase C by a Gq-type G protein.
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PMID:A Gq-type G protein couples muscarinic receptors to inositol phosphate and calcium signaling in exocrine cells from the avian salt gland. 851 32

1. In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)-dependent inhibition of contractility in rat isolated atria. 2. Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose-response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor-dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8-bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4. These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.
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PMID:Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response. 856 14


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