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)

Characterization of acetylcholine-receptor-enriched membranes from Torpedo californica electric tissue by negative-staining electron-microscopy and by lipid analysis is described. The protein/lipid ratio is 70%/30%. The lipids consist of 70% phospholipids (46% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine, 7% sphingomyelin, 2% phosphatidylinositol of the phospholipids determined) and 20% cholesterol. The acetylcholinesterase-enriched membranes show a similar composition. The only differences are a lower protein/lipid ratio (45%/55%) and a lower phosphatidylcholine/sphingomyelin ratio of 39%/14% as compared to 46%/7% for the receptor-enriched membranes. A method of preparing single-walled phosphatidylcholine vesicles by gel filtration on Sephadex G50 according to Brunner et al. (Biochem. Biophys. Acta, 455, 322--331, 1976) is used to recombine the lipid-depleted receptor complex with artificial lipid vesicles. Starting from a lipid mixture of 46% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine, 7% sphingomyelin, 2% phosphatidylinositol and 15% cholesterol we obtained vesicles associated with the acetylcholine receptor complex. These receptor vesicles are chemically excitable by 10 micrometer carbamoylcholine as measured by efflux of 22Na+ from the vesicles. The excitability is blocked by preincubation with 0.5 mM alpha-toxin from Naja naja siamensis venom and by reduction with 5 mM dithioerythritol.
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PMID:Membranes rich in acetylcholine receptor: characterization and reconstitution to excitable membranes from exogenous lipids. 63 24

The action of 1-pyrene-butyrylcholine, a new cholinergic fluorescent probe, has been studied at the cellular level using electrophysiological and fluorescence techniques. The spectroscopic properties of the probe were found to be similar to those pf pyrene-butyric acid, the excited-state lifetime in air-saturated aqueous solutions being 92 nsec. At micromolar concentrations the probe was found to exert a nondepolarizing, reversible blocking action at the neuromuscular junction of the frog. The same cholinolytic effect was observed in hypersensitive denervated muscles. The synaptic localization of the probe could be observed with fluorescence microscopy using sub- and micromolar concentrations. Treatment of the nerve-muscle preparations with proteolytic enzymes, resulting in the separation of the nerve ending from the muscle end-plate, enabled a distinction to be made between the fluorescence arising from these two parts of the synapse. Intense presynaptic fluorescence was observed, and was not altered by micromolar concentrations of alpha-bungarotoxin, d-tubocurarine, hemicholinium, or cholinesterase inhibitors. Faint reversible staining of the end-plate region was observed in enzymically treated muscles and was inhibited by prior treatment with alpha-bungarotoxin. Fluorescent alpha-toxin revealed similar patterns of fluorescence in the end-plate of enzyme-treated muscles. The postsynaptic localization of the fluorescent probe is therefore tentatively identified as the one producing the cholinolytic effect upon binding to acetylcholine receptor sites.
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PMID:1-Pyrene-butyrylcholine: a fluorescent probe for the cholinergic system. 108 Dec 27

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.
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PMID:Acetylcholinesterases of the nematode Steinernema carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association. 132 59

The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.
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PMID:Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C. 132 66

In Torpedo electric organ, much of the acetylcholinesterase (AChE) is a globular dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and selectively solubilized by a bacterial phosphatidylinositol-specific phospholipase C. While the structure of this form of the enzyme is well-established, the ultrastructural localization of G2-AChE is still unclear. Selective solubilization with phosphatidylinositol-specific phospholipase C was, therefore, combined with immunocytochemistry at the electron microscope level, in order to localize G2-AChE in electric organ of Torpedo ocellata. Thin sections of electric organ were labelled with antibodies raised against Torpedo AChE, followed by gold-conjugated second antibodies, before or after exposure to the phospholipase. For comparison, the location of AChE was examined using histochemical methods. We show that (1) immunolabelling is concentrated in the synaptic clefts between nerve terminals and the innervated face of the electrocyte; (2) this labelling co-localizes with AChE histochemical reaction products; and (3) prior exposure to the phospholipase causes a decrease in AChE-associated labelling. Quantitative analysis of immunolabelling in the synaptic clefts shows that the phospholipase treatment had reduced primary labelling at or adjacent to the presynaptic membrane. Together with our earlier biochemical and immunofluorescent evidence, these results support our previous assignment of a neuronal and synaptic localization for G2-AChE in Torpedo electric organ.
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PMID:G2-acetylcholinesterase is presynaptically localized in Torpedo electric organ. 133 40

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.
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PMID:Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid. 138 56

Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5-4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes.
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PMID:Alkylacyl glycerophosphoinositol in human and bovine erythrocytes. Molecular species composition and comparison with glycosyl-inositolphospholipid anchors of erythrocyte acetylcholinesterases. 139 75

The properties of a cholinesterase from mucosal cells of rat intestine have been characterized. The enzyme was identified as butyrylcholinesterase because it was more sensitive to iso-OMPA (IC50 = 1.0 x 10(-6) M) than to BW284C51 (IC50 = 5.5 x 10(-5) M) and was not inhibited by substrate excess. It displayed a higher affinity for acetylthiocholine than for butyrylthiocholine. A major molecular form was observed sedimenting at 5.9 S. Two other minor molecular forms were identified as a hydrophilic tetramer (G4, sedimenting at 10.5 S) and a monomer (G1, sedimenting at 4.3 S). The 5.9 S component was referred to as "G" form (G for globular) and not "G2" as usual dimers for the following reasons: (i) the G form was unaffected by the reducing agents, beta-mercaptoethanol and dithiothreitol, which converted disulfide-linked dimers of acetylcholinesterase into monomers, (ii) the G form was shifted from 5.9 to 3.4 S when the sucrose gradient contained Triton X-100. This value of 3.4 S (in Triton X-100) appeared too low for a typical G2 form. The shift in the S value was partly reversible: the 3.4 S form resedimented at 5.2 S in the absence of detergent. The behavior of the G form in sucrose gradients indicated that it was amphiphilic. This was confirmed in nondenaturing electrophoreses and also by quantitative binding of the G form to octyl-Sepharose. The hydrophobic domain of the G form was not a glycolipid, as shown by its insensitivity to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and its nonaggregating properties in the absence of nondenaturing detergent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amphiphilic forms of butyrylcholinesterase in mucosal cells of rat intestine. 142 Feb 1

Both salt-soluble and detergent-soluble rat brain globular acetylcholinesterases (SS- and DS- AChE EC 3.1.1.7) are amphiphiles, as shown by detergent dependency of enzymatic activity and binding to liposomes. Proteinase K and papain treatment transformed SS-AChE and DS-AChE into forms that, in absence of detergent, no longer aggregated nor bound to liposomes. In contrast, phosphatidylinositol-specific phospholipase C had no effect on these properties. Labeling DS-AChE with 3-(trifluoromethyl)-3-(m-(125I)-iodophenyl) diazirine ([125I]TID) revealed, by polyacrylamide gel electrophoresis under reducing conditions, one single band of 69 kD apparent molecular mass. The same pattern was previously obtained with Bolton and Hunter reagent-labeled enzyme. Proteinase K treatment transformed the 11 S [125I]TID labeled AChE into a 4 S form which no longer showed 125I-radioactivity and was unable to bind to liposomes. These results are compatible with the existence of a hydrophobic segment present both on salt-soluble and detergent-soluble 11 S AChE as well as on the minor forms 4 S and 7 S. This segment is not linked to the catalytic subunits by disulfide bounds in contrast to the 20 kD non-catalytic subunit described by Inestrosa et al.
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PMID:A unique hydrophobic domain of rat brain globular acetylcholinesterase for binding to cell membranes. 146 72

Membrane-bound acetylcholinesterase (AChE) from the human erythrocyte is inhibited by chlorpromazine (CPZ) in a concentration range within this amphiphilic drug has been demonstrated to interact with erythrocyte membranes, causing a large spectrum of physical and structural effects; membrane solubilization with 0.5% Triton X-100 results in a complete loss of CPZ inhibitory potency. Although these observations might suggest a role of membrane lipid environment in mediating human erythrocyte AChE inhibition, we observed that CPZ retains its full inhibitory effect on the fraction of enzyme (5-6% of total) that is solubilized from erythrocytes upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis; furthermore, Triton X-100 is able to reverse the CPZ effect also in the case of PI-PLC-solubilized enzyme. These results demonstrate unequivocally that CPZ inhibits human erythrocyte AChE through direct molecular interaction. The inhibition kinetics displayed by CPZ on human erythrocyte AChE are dependent on drug concentration: evidence is provided that this phenomenon may be related to formation of CPZ micellar aggregates.
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PMID:A study of human erythrocyte acetylcholinesterase inhibition by chlorpromazine. 165 84


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