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)

After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent Mr 41,000, 50,000, and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of Mr 43,000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of 30 micron carbamoylcholine to occupy acetylcholine binding sites, [14C]-meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide ] was bound with a dissociation constant, KD, of 0.3 +/- 0.1 micron to 0.3 +/- 0.1 site per [3H]alpha-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.
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PMID:Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. 3 54

1. When heated in 8 M-urea, phospholipase C(EC 3.1.4.3) from Bacillus cereus undergoes conformational transitions depending on the temperatures used. These transitions were studied by examining protein fluorescence, iodide quenching of protein fluorescence, u.v. difference spectroscopy, chemical availability of histidine residues in the enzyme, circular dichroism and catalytic activity. 2. Unless simultaneously exposed to elevated temperatures the enzyme appears to be unaffected by 8 M-urea. Removal of the two zinc atoms from the enzyme renders phospholipase C very sensitive to denaturation by 8 M-urea as indicated by fluorescence emission spectra and circular dichroism. 3. Both the native and the zinc-free enzymes are markedly more resistant to irreversible thermal inactivation in the presence of 8 M-urea than in its absence. 4. The response of the enzyme to 8 M-urea and the role of zinc in stabilizing the enzyme are discussed.
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PMID:Conformational studies on phospholipase C from Bacillus cereus. The effect of urea on the enzyme. 10 29

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.
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PMID:Iodination of a tyrosyl residue in staphylococcal alpha-toxin. 127 41

The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.
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PMID:Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue. 159 35

FRTL-5 thyroid cells express a muscarinic receptor which inhibits the phospholipase C activity in a pirenzepine-insensitive manner. We here report that the cholinergic agonist carbachol decreases in these cells the steady-state iodide content, an effect correlated with the iodination of thyroglobulin and with thyroid hormone formation. Several signal pathways may be involved in this phenomenon since carbachol in addition to inhibiting phospholipase C, increased the arachidonic acid release and modified the adenylyl cyclase activity. In FRTL-5 cells, arachidonic acid is released via the direct stimulation of phospholipase A2 by a pirenzepine-sensitive muscarinic receptor coupled to a GTP binding protein sensitive to pertussis toxin. Regarding adenylyl cyclase, carbachol potentiated the thyrotropin-induced stimulation of the enzyme, whereas it did not affect the basal levels of cAMP. In vitro binding studies revealed the presence of two muscarinic binding sites. To summarize, the analysis of signal pathways and of in vitro binding sites indicates a complex muscarinic regulation of thyroid function, which includes the modulation of iodide fluxes.
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PMID:Muscarinic regulation of phospholipase A2 and iodide fluxes in FRTL-5 thyroid cells. 165 22

Tumor-promoting phorbol esters, e.g., 12-O-tetradecanoylphorbol 13-acetate (TPA), inhibit TSH-stimulated iodide organification in vitro implying a role for protein kinase C (PKC) in the regulation of differentiated thyroid function. To further explore the PKC dependence of this action of TPA, we studied the effects of PKC inhibition and downregulation on phorbol-mediated differentiated thyroid function in vitro. In addition, the effects of the nonphorbol PKC activator, phospholipase C (PLC) were studied. TPA (100 nM) inhibited TSH-stimulated iodide organification in cultured porcine thyroid cells by over 95% and caused PKC translocation in vitro. Exogenous PLC (1 U/mL) could mimic these effects of TPA. The PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) inhibited TSH-stimulated iodide organification at concentrations exceeding 10 microM. However, partial recovery of phorbol- and PLC-inhibited iodide organification was seen in the presence of identical concentrations of H7. H7 had no effect on PKC translocation in porcine thyroid cell extracts. After 24 h of TPA treatment to induce PKC downregulation, no recovery of TSH-stimulated iodide organification was observed, suggesting that the effects of TPA were irreversible. These studies indicate that the effects of TPA and PLC on differentiated thyroid function are mediated, at least in part, by PKC. These findings provide further evidence for a role for PKC in the regulation of differentiated thyroid function.
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PMID:Phorbol ester and phospholipase C-mediated differentiated thyroid function in vitro: the effects of protein kinase C inhibition and downregulation. 182 67

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.
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PMID:Manoalide, a natural sesterterpenoid that inhibits calcium channels. 243 21

The action of carbamoylcholine (Cchol), NaF and other agonists on the generation of inositol phosphates (IPs) was studied in dog thyroid slices prelabelled with myo-[2-3H]inositol. The stimulation by Cchol (0.1 microM-0.1 mM) of IPs accumulation through activation of a muscarinic receptor [Graff, Mockel, Laurent, Erneux & Dumont (1987) FEBS Lett. 210, 204-210] was pertussis- and cholera-toxin insensitive. Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4 were generated. NaF (5-20 mM) also increased IPs generation (Graff et al., 1987); this effect was potentiated by AlCl3 (10 microM) and unaffected by pertussis toxin. Although phorbol dibutyrate (5 microM) abolished the cholinergic stimulation of IPs generation (Graff et al., 1987), it did not affect the fluoride-induced response. Cchol and NaF did not require extracellular Ca2+ to exert their effect, and neither KCl-induced membrane depolarization nor ionophore A23187 (10 microM) had any influence on basal IPs levels, or on cholinergic stimulation. However, more stringent Ca2+ depletion with EGTA (0.1 or 1 mM) decreased basal IPs levels as well as the amplitude of the stimulation by Cchol without abolishing it. Dibutyryl cyclic AMP, forskolin, cholera toxin and prostaglandin E1 had no effect on basal IPs levels and did not decrease the response to Cchol. Iodide (4 or 40 microM) also strongly decreased the cholinergic action on IPs, this inhibition being relieved by methimazole (1 mM). Our data suggest that Cchol activates a phospholipase C hydrolysing PtdIns(4,5)P2 in the dog thyroid cell in a cyclic AMP-independent manner. This activation requires no extracellular Ca2+ and depends on a GTP-binding protein insensitive to both cholera toxin and requires no extracellular Ca2+ and depends on a GTP-binding protein insensitive to both cholera toxin and pertussis toxin. The data are consistent with a rapid metabolism of Ins(1,4,5)P3 to Ins(1,3,4)P3 via the Ins(1,4,5)P3 3-kinase pathway, followed by dephosphorylation by a 5-phosphomonoesterase. Indeed, a Ca2+-sensitive InsP3 3-kinase activity was demonstrated in tissue homogenate. Stimulation of protein kinase C and an organified form of iodine inhibit the Cchol-induced IPs generation. The negative feedback of activated protein kinase C could be exerted at the level of the receptor or of the receptor-G-protein interaction.
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PMID:Stimulation of generation of inositol phosphates by carbamoylcholine and its inhibition by phorbol esters and iodide in dog thyroid cells. 255 11

Radioligand binding and ESR were used to study the association of a spin-labeled local anesthetic, 2-[N,N-dimethyl-N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-4-hexyloxybenzoate iodide (C6SLMel), with acetylcholine receptor-enriched membranes from Torpedo californica. In the presence of carbamylcholine, we found that C6SLMel competitively inhibits [3H]phencyclidine binding with high affinity (KD = 8.7 X 10(-7) M for C6SLMel), whereas in the presence of alpha-toxin or in the absence of agonist, C6SLMel binds with lower affinity (KD = 2 X 10(-5) M). At concentrations lower than 1 X 10(-5) M, C6SLMel does not bind to the agonist site but enhances [3H]acetylcholine binding. These findings show that C6SLMel binds to the allosterically coupled noncompetitive inhibitor site which is regulated by agonist binding. In addition, C6SLMel preferentially associates with the desensitized receptor state known to exhibit high affinities for agonists and local anesthetics. ESR measurements of C6SLMel bound to receptor membranes in the absence of agonist show moderately immobilized spectra. Addition of carbamylcholine results in the appearance of a strongly immobilized component. Prior exposure to alpha-toxin blocks the carbamylcholine-induced, strongly immobilized component in the ESR spectrum. Furthermore, in the presence of carbamylcholine, back-titration of bound C6SLMel with phencyclidine decreases the highly immobilized component at concentrations consistent with the KD for phencyclidine. These findings indicate that C6SLMel detects conformational changes between the resting and desensitized acetylcholine receptor states that occur at the noncompetitive inhibitor binding site. The strongly mobilized component is not affected by ferricyanide addition, suggesting that the binding site is in a region not readily accessible to anion collision from the aqueous phase.
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PMID:Association of a spin-labeled local anesthetic with the allosterically coupled noncompetitive inhibitor site on the acetylcholine receptor. 301 81

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.
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PMID:Cholinergic stimulation of inositol phosphate formation in bovine adrenal chromaffin cells: distinct nicotinic and muscarinic mechanisms. 366 43


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