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)

Binding of acetylcholine in the concentration range 1 nM-1 muM was measured by equilibrium dialysis to a particulate preparation of Torpedo electroplax, without or with prior treatment of the tissue with one of three chemical modifying reagents. Significant reduction in binding of acetylcholine resulted after treatment with 1,4-dithiothreitol, p-chloromercuribenzoate, or p-(trimethylammonium)-benzenediazonium fluoroborate. Partial reversal of the reduction in binding occurred when dialysis was performed in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) or potassium ferricyanide (in the case of treatment with dithiothreitol), and 2-mercaptoethanol (in the case of treatment with p-chloromercuribenzoate). It is concluded that the functional acetylcholine-receptor macromolecule of Torpedo electroplax has disulfide bond(s), sulfhydryl group(s), and one or more of the amino acids vulnerable to diazotization by p-(trimethylammonium)-benzenediazonium fluoroborate. This, plus the effect of phospholipase C (EC 3.1.4.3) in elimination of detectable binding of acetylcholine after electrofocusing, is additional evidence that the functional acetylcholine receptor is a phospholipoprotein or a phospholipid-protein complex, which has a low isoelectric point of 4.5 +/- 0.2, yet is denatured by exposure to low pH for 24 hr. Due to this adverse effect, recovery of binding of acetylcholine after electrofocusing, as detected by equilibrium dialysis or ultrafiltration, is only 23% and, so far, only 6.3-fold purification of functional acetylcholine receptors by this technique is possible. Three or two forms of acetylcholinesterase (EC 3.1.1.7), whose peaks have isoelectric points ranging from 4.3 to 7.2, appear after electrofocusing of Torpedo extracted with 1% Triton X-100 or Lubrol, respectively. The major peak in either preparation has an isoelectric point of 5. Although the peaks of the functional acetylcholine receptors and of acetylcholinesterase of Torpedo electroplax are separable by electrofocusing, it has not been possible to isolate fractions that contain functional receptors but that are free of acetylcholinesterase. The opposite is possible.
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PMID:Characterization and partial purification of the acetylcholine receptor from Torpedo electroplax. 450 55

Purified alpha-toxin from Naja nigricollis snake venom labeled by [(3)H]acetylation binds specifically to the acetylcholine receptors of mouse neuroblastoma cells. Toxin binding was inhibited by inhibitors for nicotinic and muscarinic acetylcholine receptors. Clones of neuroblastoma cells were selected for low acetylcholinesterase (EC 3.1.1.7) activity with antibodies against this enzyme. Selection for an 80-fold decrease in acetylcholinesterase activity was not associated with any decrease in the number of acetylcholine receptors (3.4 x 10(7) per cell). Removal or inactivation of 80% of the acetylcholine receptors by proteolytic enzymes or by compounds that block sulfhydryl groups did not change the activity of acetylcholinesterase on the cell surface. In addition to these results on the separation between acetylcholine receptors and acetylcholinesterase, a common regulation was found in that both the number of acetylcholine receptors and the activity of acetylcholinesterase were increased 5- to 10-fold when the cells stopped to multiply or were induced to differentiate by dibutyryl-cyclic AMP. It is suggested that there are different genes for the acetylcholine receptor and acetylcholinesterase, and that both are regulated during growth and differentiation by a common regulatory gene.
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PMID:Regulation of acetylcholine receptors in relation to acetylcholinesterase in neuroblastoma cells. 451 44

An equilibrium dialysis technique, applied to lyophilized particulate fractions of Torpedo electroplax, gave data consistent with a single kind of macromolecular binding of muscarone, with binding constant, 7 x 10(-7)M and an amount of 1 nmole per gram original electroplax. The effects on muscarone binding of 38 drugs suggested that muscarone binding reflected acetylcholine receptor activity. Of 18 enzyme preparations, only trypsin, chymotrypsin, and phospholipase C reduced binding activity, suggesting that a phospholipoprotein was binding. Partial "solubilization" of the binding protein was achieved, but the "solubilized" activity did not migrate on electrophoresis. Additional evidence was provided that acetylcholinesterase was not responsible for this muscarone binding.
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PMID:A muscarone-binding material in electroplax and its relation to the acetylcholine receptor. II. Dialysis assay. 526 75

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the alpha-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with N-ethylmaleimide and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins (6.0 +/- 0.6 MSL molecules per alpha-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving 2.2 +/- 0.4 MSL molecules per alpha-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with N-[14C] ethylmaleimide suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (rotational correlation time tau C greater than 1 ms). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane (100 less than or equal to tau c less than or equal to 120 microseconds). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-proteins ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor and rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored (tau c greater than or equal to 1 ms).
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PMID:Effects of temperature, lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmarata. 627 50

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na+ + K+)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C12E8). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K+-phosphatase or (Na+ + K+)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the alpha- and beta-subunits. The (Na+ + K+)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis alpha-toxin-Sepharose columns. The data suggest that the alpha-subunit of (Na+ + K+)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.
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PMID:Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata. 629 54

We have studied the kinetics of N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin (FITC-toxin) binding to the membrane-associated acetylcholine receptor from the Torpedo californica electric organ. The fluorescent toxin not only enabled us to monitor the binding reaction continuously but also to examine simultaneously the enhancement of ligand fluorescence and the increase in steady state polarization of fluorescence associated with binding of the alpha-toxin. Over the range of concentrations employed, both parameters yielded identical kinetic constants, suggesting that the enhancement of fluorescence of fluorescein and its immobilization are occurring in the same time frame. Both an initial rate analysis and the integrated rate expression showed association to be a simple, reversible bimolecular process. The apparent second-order association rate constant derived from the integrated rate analysis was constant within a factor of 2 over a 40-fold concentration range (6.7 +/- 1.7 X 10(3) M-1 S-1). The unimolecular dissociation rate constant was found to be 3.3 +/- 0.5 X 10(-5) S-1.
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PMID:Kinetics of interaction of N epsilon-fluorescein isothiocyanate-lysine-23-cobra alpha-toxin with the acetylcholine receptor. 669 70

An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins bind to the two alpha-toxin-binding sites, we derived an equation which allows prediction of a "true" efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching. Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled alpha-toxins, N epsilon-fluorescein isothiocyanate lysine 23 alpha-toxin and monolabeled tetramethylrhodamine isothiocyanate alpha-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.
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PMID:Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor. 671 68

Cobra alpha-toxin purified from Naja naja siamensis venom was labeled with near stoichiometric quantities of fluorescein isothiocyanate. A monofluorescein alpha-toxin was separated in 50-60% yield from unconjugated alpha-toxin and other reaction products by ion exchange chromatography. The isolated mono-conjugated alpha-toxin electrofocuses largely as a single entity with 92% appearing with a pI of 9.6. The unmodified toxin has a pI of 10.7. Thermolysin digestion and subsequent high pressure liquid chromatography of the peptides yield two dominant fluorescent peaks, both of which can be traced to the labeling of lysine 23. The NE-fluorescein isothiocyanate (FITC)-Lys-23 alpha-toxin shows an apparent reduction in quantum yield when compared with either free FITC or the denatured and reduced NE-FITC-Lys-23 alpha-toxin. The reduction of fluorescence is likely to be due to static quenching of the fluorescein by the tryptophanyl and tyrosyl residues (25 and 21, respectively) in the "central loop" region. Binding of the NE-FITC-Lys-23 alpha-toxin to the membrane-associated acetylcholine receptor is accompanied by a 95 +/- 22% increase in fluorescence which probably reflects perturbation of the beta-pleated sheet character of the region containing residues 20-25 of the alpha-toxin. Steady state fluorescence polarization measurements of NE-FITC-Lys-23 alpha-toxin yield a rotational correlation time of 3.7 ns, suggesting FITC is largely immobilized on the alpha-toxin. The NE-FITC-Lys-23 alpha-toxin binds with a dissociation constant of 4 nM determined by fluorescence polarization.
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PMID:Site-specific fluorescein-labeled cobra alpha-toxin. Biochemical and spectroscopic characterization. 680 19

3H chlorpromazine binds to acetylcholine receptor-rich membrane fragments prepared from Torpedo marmorata electric organ in three different manners: (1) at the level of high affinity sites from which it is displaced by perhydrohistrionicotoxin, (2) at the level of low affinity sites insensitive to this toxin, and (3) in a non saturable manner to a presumably lipidic phase. Binding of chlorpromazine to the first two categories of sites independently stabilizes the "desensitized" high affinity state of the receptor for cholinergic agonists. There exists one high affinity site per two snake alpha-toxin sites, thus per 250,000 daltons light form of the receptor. Under the conditions of 3H chlorpromazine high affinity binding, ultraviolet irradiation results in the covalent incorporation of this ligand to the four chains of the receptor.
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PMID:[The high affinity binding site for chlorpromazine is present only as a single copy per cholinergic receptor molecule and is shared by four polypeptide chains]. 681 70

Reversible ligands were attached covalently to membrane-bound acetylcholine receptor from Torpedo marmorata by a method which is generally applicable and does not require the synthesis of specially designed molecules. UV irradiation of the receptor in the presence of [3H]trimethisoquin, [3H]phencyclidine, or [3H]perhydrohistrionicotoxin resulted in the labeling of the binding site(s) for these noncompetitive blockers of the permeability response. The labeling of the delta chain was enhanced by carbamoylcholine, and this increase was blocked by snake alpha-toxins. The effect of carbamoylcholine on [3H]trimethisoquin binding was more pronounced than with the other two noncompetitive blockers; in all instances, the labeling was abolished by unlabeled histrionicotoxin. These three compounds therefore interact with the high-affinity site for noncompetitive blockers. Incorporation of radioactivity also occurred into the alpha chain but either was insensitive to cholinergic effectors or decreased in the presence of carbamoylcholine (or snake alpha-toxin), probably as a result of an interaction with the acetylcholine-binding site. In contrast to the other noncompetitive blockers tested, [3H]chlorpromazine heavily labeled the four receptor polypeptides (alpha, beta, gamma, delta), and this labeling also was enhanced by carbamoylcholine and decreased by histrionicotoxin. These data indicate a contribution of the delta chain to the binding site(s) of several well-characterized noncompetitive blockers and suggest that other receptor polypeptides may also contribute to this binding.
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PMID:Ultraviolet light-induced labeling by noncompetitive blockers of the acetylcholine receptor from Torpedo marmorata. 694 90


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