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 various sugars was compared in purified subfractions of taste buds isolated from bovine circumvallate papillae and of non-taste bud-bearing epithelium isolated from tissue surrounding these papillae. Binding of 14C-labeled sugars was greater in purified subfractions obtained from taste bud than from non-taste bud-bearing tissue and was, in general, greater in those taste bud subfractions in which a greater membrane purification was achieved. Binding specificity of the 14C-labeled sugars sucrose, fructose, glucose and of 14C-labeled cyclamate and saccharine was measured by competition of each 14C-labeled sugar or synthetic sweetener with its unlabeled homologous sugar in P4(B) taste bud subfractions; this binding, as shown for sucrose, was reversible and temperature dependent. Essentially no competition of the 14C-lageled sugars sucrose, fructose, glucose or 14C-labeled cyclamate and saccharine by their respective unlabeled homologues occurred in epithelial tissue P4(B) subfractions; this binding was not reversible. Binding specificity was further observed by the competition of 14C-labeled sucrose, fructose and glucose with each unlabeled sugar for binding sites on P4(B) taste bud subfractions; unlabeled sucrose was more effective in competing with each 14C-labeled surgar than was unlabeled fructose or glucose. The relatively non-sweet sugar lactose did not compete with 14C-labeled lactose in P4(B) subfractions from either taste bud or non-taste bud-bearing epithelial tissue. Binding of 14C-labeled sucrose in purified P4(B) bud subfractions was inhibited by increased concentrations of unlabeled sucrose, phospholipase C, neuraminidase, EDTA, NaCl and urea. Dissociation constants for sugar or synthetic sweetener binding were low (approx. 10(-3) M) but in a rank order (sucrose greater than fructose greater than glucose greater than saccharine) consistent with preference and electrophysiological responses in cow. The cow is behaviorally indifferent to saccharine and lactose consistent with the data obtained in the present study.
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PMID:Sugar binding to purified fractions from bovine taste buds and epithelial tissue. Relationships to bioactivity. 81 29

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.
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PMID:Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton. 142 46

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and mechanism of alkaline phosphatase. 152 73

A model of transducin activation is constructed from its partial reactions (formation of metarhodopsin II, association, and dissociation of the rhodopsin-transducin complex). The kinetic equations of the model are solved both numerically and, for small photoactivation, analytically. From data on the partial reactions in vitro, rate and activation energy profile of amplified transducin turnover are modeled and compared with measured light-scattering signals of transducin activation in intact retinal rods. The data leave one free parameter, the rate of association between transducin and rhodopsin. Best fit is achieved for an activation energy of 35 kJ/mol, indicating lateral membrane diffusion of the proteins as its main determinant. The absolute value of the association rate is discussed in terms of the success of collisions to form the catalytic complex. It is greater than 30% for the intact retina and 10 times lower after permeabilization with staphylococcus aureus alpha-toxin. Dissociation rates for micromolar guanosinetriphosphale (GTP) (Kohl, B., and K. P. Hofmann, 1987. Biophys. J. 52:271-277) must be extrapolated linearly up to the millimolar range to explain the rapid transducin turnover in situ. This is interpreted by an unstable rhodopsin-transducin-GTP transient state. At the time of maximal turnover after a flash, the rate of activation is determined as 30, 120, 800, 2,500, and 4,000 activated transducins per photoactivated rhodopsin and second at 5, 10, 20, 30, 37 degrees C, respectively.
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PMID:Reaction rate and collisional efficiency of the rhodopsin-transducin system in intact retinal rods. 190 Dec 31

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.
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PMID:The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems. 213 29

The characteristics of the specific binding of labelled insulin to turtle thyroid microsomes were investigated. Binding experiments were performed in Krebs-Ringer bicarbonate buffer (pH 7.4) at 25 or 4 degrees C for different periods of time. Dissociation of the labelled insulin from the binding sites was also evaluated. It was found that the binding is dependent on time, temperature and microsomal protein concentration, with an optimum pH of 8.0. Unlabelled insulin and pro-insulin competed with the labelled insulin, binding in direct proportion to their biological activities, while glucagon and growth hormones did not compete for the binding sites. Scatchard plot analysis established the presence of binding sites of high and low affinities, and the rate of dissociation of bound insulin was considerably increased by the addition of unlabelled insulin. Both results are compatible with a negative co-operativity site-site interaction model. Trypsin abolished the insulin binding. These findings indicate that the microsomes from the turtle thyroid gland contain specific binding sites for insulin. However, pre-incubation of microsomes with phospholipase C or S-adenosyl-L-methionine (SAM), or incubation in the presence of 2 mol NaCl/l did not increase the specific insulin binding. Therefore, the binding properties are similar to those observed in mammalian insulin-responsive tissues except for the absence of the effects of 2 mol NaCl/l, phospholipase C or SAM, which suggests the absence of masked insulin-binding sites.
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PMID:Characterization of the insulin-binding sites in turtle thyroid microsomes. 351 Nov 68

The effects of tumour-promoting phorbol esters on the receptor-mediated endocytosis of insulin were investigated in the human hepatoma cell line HepG2. Treatment of these cells with the biologically active phorbol 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with the non-tumour-promoting analogue 4 alpha-phorbol 12,13-didecanoate, resulted in dramatic morphological changes, which were accompanied by a 1.5-2.5-fold increase in specific 125I-insulin association with the cells at 37 degrees C. This increase in insulin binding was not observed when the binding reaction was performed at 4 degrees C. The potentiation of 125I-insulin association with TPA-treated cells at 37 degrees C could be completely accounted for by an increase in the intracellular pool of internalized insulin; there was no concomitant increase in cell-surface insulin binding. Dissociation studies showed that the enhanced internalization of insulin by cells after treatment with TPA resulted from a decrease in the rate of intracellular processing of the insulin after receptor-mediated endocytosis. The phorbol-ester-induced enhancement of internalized insulin in HepG2 cells was additive with the potentiation of endocytosed insulin induced by both the lysosomotropic reagent chloroquine and the ionophore monensin; this indicates that TPA affects the intracellular processing of the insulin receptor at a point other than those disrupted by either of these two reagents. The potentiation of insulin receptor internalization by tumour-promoting phorbol esters could be completely mimicked by treatment with phospholipase C, but not with phospholipase A, and partially mimicked by treatment with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol. By these criteria, the effects of phorbol esters on the insulin receptor in HepG2 cells appear to be mediated through protein kinase C. These results support the concept that the activation of protein kinase C by treatment with phorbol esters causes a perturbation of the insulin-receptor-mediated endocytotic pathway in HepG2 cells, reflected in a long-term decreased rate of dissociation of internalized insulin by the phorbol-ester-treated cells.
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PMID:Potentiation of specific association of insulin with HepG2 cells by phorbol esters. 353 1

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.
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PMID:Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor. 365 51

Evaluation of TSH binding to plasma membranes of porcine thyroid revealed unique sensitivity to pH and temperature. Analysis of apparent equilibrium binding yielded a linear Scatchard plot at the optimal pH of 6.0, indicating one class of binding sites. At physiological pH 7.4 a curvilinear Scatchard plot was obtained, resolved by computer analysis into two classes of binding sites of different affinities and capacities. Treatment of membranes with phospholipase C resulted in a 20% decrease in the number of high affinity sites, but no change occurred in binding affinity. In contrast, low affinity sites were not altered. To evaluate the significance of the curvilinear Scatchard plot, the kinetics of association were examined. The intrinsic Kd (kd/ka) was 0.20 nM, a value essentially equivalent to that of the high affinity binding component. The 'negative cooperativity' model of hormone binding was evaluated by examining the effect of excess unlabeled TSH on dissociation rate. Dissociation of bound 125I-labeled TSH was biphasic, and was enhanced by unlabeled hormone, regardless of whether the membranes were prelabeled at pH 6.0 or 7.4. This effect was not correlated with curvilinear Scatchard plots, and therefore not proof of negative cooperativity. Binding sites for TSH were further distinguished by their sensitivity to temperature. A van't Hoff plot of temperature dependence of the apparent Kd of the high affinity site was linear from 4 to 37 degrees C. In contrast, the apparent Kd of low affinity binding did not vary with respect to temperature. These results demonstrate that there are at least two independent binding sites for TSH on porcine thyroid plasma membranes, distinguishable by their equilibrium binding properties.
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PMID:Thyrotropin binding to porcine thyroid plasma membranes: kinetic and thermodynamic analyses. 715 94

Src homology regions 2 (SH2) and 3 (SH3) are noncatalytic domains that are conserved among several proteins implicated in the regulation of cell proliferation. Using bacterially expressed fusion proteins containing the SH2 domain of the abl tyrosine kinase, we have quantitated the binding of these domains to the activated epidermal growth factor (EGF) receptor (EGFR). A 35S-labeled abl SH2 fusion protein binds to the human EGFR immunoprecipitated from EGF-treated NIH3T3 cells that overexpress the receptor. This binding is totally dependent on the pretreatment of cells with EGF. The interaction is rapid, reaching 50% of maximum within 1 min, and attaining apparent equilibrium by 10 min. Dissociation of the complex is biphasic with a rapidly dissociating component (t1/2 of less than 1 min), as well as a slowly dissociable component. The 35S-labeled abl SH2 fusion protein specifically binds to the EGFR in a saturable manner and is differentially inhibited by unlabeled fusion proteins containing SH2 domains from phospholipase C, the p85 subunit of phosphatidylinositol-3 kinase, and the GTPase activation protein of ras. To identify residues critical for abl SH2-EGFR binding, six point mutants were constructed in the highly conserved FLVRES motif. Three mutants (V170L, E172Q, and E174Q) display binding affinities similar to that of wild type. However, three other mutants (R171K, S173C, and S175C) have greatly reduced affinity. Interestingly, the binding affinity to the EGFR determined by the in vitro assay directly correlates with the transforming ability of the corresponding v-abl constructs in vivo (Mayer, B. J., Jackson, P. K., Etten, R. A. V., and Baltimore, D. (1992) Mol. Cell. Biol. 12, 609-618). These data indicate that the Arg-171, Ser-173, and Ser-175 are critical for both transformation and abl SH2 domain binding to phosphotyrosine-containing proteins.
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PMID:Direct analysis of the binding of the abl Src homology 2 domain to the activated epidermal growth factor receptor. 767 9


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