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)

Ornithine decarboxylase has been identified and characterized in the free-living nematode Caenorhabditis elegans. Unlike previously described ornithine decarboxylases, the enzyme activity is membrane-associated and remains in the membrane fraction after treatment with high salt, detergents or phosphatidylinositol-specific phospholipase C. Ornithine has an apparent Km value of 2.7 microM for ornithine decarboxylase. The enzyme is competitively inhibited by arginine and lysine with Ki values of 4.0 and 24.4 microM respectively. None of the other naturally occurring amino acids inhibited more than 10% of the enzyme activity at concentrations up to 1 mM. Agmatine, putrescine, spermidine and spermine inhibit ornithine decarboxylase in a non-competitive manner with Ki values of 10, 53.5, 59 and 855 microM respectively. A similar ornithine decarboxylase activity was also identified in membrane preparations from the parasitic nematode Haemonchus contortus.
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PMID:Characterization of a high-affinity membrane-associated ornithine decarboxylase from the free-living nematode Caenorhabditis elegans. 224 95

Investigations on the turnover of the membrane-form variant surface glycoprotein (mfVSG) of Trypanosoma brucei during cultivation in vitro of the monomorphic variant clones MIT at 1.2 and MIT at 1.4 showed that bloodstream forms slowly released the surface coat into the medium (time required to decline to half the initial amount, t50% = 32 +/- 3 h). VSG appeared in the medium in its soluble form (sVSG) which lacked the dimyristoylglycerol membrane anchor as judged by electrophoretic mobility and exposure of the cross-reacting determinant. The total VSG in the culture was very stable with a t50% = 189 +/- 24 h, compared to the other cellular proteins with a t50% approximately 28 h. Coat release during differentiation of bloodstream forms to procyclic cells could be distinguished from this turnover both by its more rapid kinetics (t50% = 13 +/- 1 h) and by the appearance in the medium of a predominant proteolytic fragment in addition to sVSG. Coat release during the transition to procyclic forms was not inhibited by the lysosomotropic agents ammonium chloride or chloroquine, by the proton ionophore monensin, or by the protease inhibitor tosyl-L-lysine chloromethyl ketone. The experiments demonstrate that coat release during differentiation is a specific cellular event distinct from simple turnover. The possibility is discussed that VSG release under both conditions occurs by endocytosis of mfVSG, degradation by a phospholipase C or a protease or both in a non-acidic intracellular compartment and recycling to the surface by exocytosis.
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PMID:Release of the variant surface glycoprotein during differentiation of bloodstream to procyclic forms of Trypanosoma brucei. 291 Dec 81

The exposure of WRK1 cells to arginine vasopressin (AVP), lysine vasopressin, or oxytocin for 18 h at 37 degrees C induced a homologous desensitization of the vasopressin- (VP) receptors. Dose-response curves of [3H]lysine vasopressin binding to control and desensitized WRK1 cells revealed a decrease in the maximal number of binding sites without any modification of its affinity (Kd values = 4.40 +/- 0.76 nM and 4.65 +/- 0.78 nM for control and desensitized conditions, respectively). The phenomenon was time- and dose-dependent. It was directly related to receptor occupancy, since the concentration of VP analogues leading to a half-maximal occupancy of VP receptors was closely related to the concentration of the corresponding analogue leading to a half-maximal decrease in VP-binding sites. It was also agonist-specific, since the V1 vasopressin antagonist desGly9d(CH2)5[D-Tyr(Et)2]VAVP was unable to affect the number of receptors. These desensitization processes were completely inhibited when the functional coated pits present in WRK1 cells were suppressed, indicating that the loss of VP-binding sites was related to receptor internalization. The exposure of WRK1 cells to a vasopressin agonist for 18 h also led to an inhibition of the vasopressin-sensitive phospholipase C activity. It was time- and agonist-dose-dependent, and occurred without any detectable changes in apparent affinity values (1.40 +/- 0.04 and 1.90 +/- 0.36 nM for control and desensitized cells, respectively). Control experiments showed that these inhibitions could not have been caused by a decrease in the labeling of inositol lipids. It is likely that they were mainly due to receptor internalization since (i) the hormonal treatment did not modify the basal level of phospholipase C; (ii) the maximal loss of VP-binding site was similar to the maximal inhibition of VP-stimulated IP accumulation; (iii) the recoveries of both VP-binding sites and VP-sensitive phospholipase C activity followed exactly the same time course (t1/2 = 4 h). In addition to this homologous desensitization of VP-sensitive phospholipase C activity, AVP also induced heterologous desensitization of bradykinin-sensitive phospholipase C activity. However, this effect was relatively weak (maximal inhibition 17 +/- 3%). The time course of VP-sensitive phospholipase C desensitization was more rapid than that of VP-receptors, indicating that desensitization involved at least two distinct steps, a rapid uncoupling step, and a later loss of vasopressin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evidence of two steps in the homologous desensitization of vasopressin-sensitive phospholipase C in WRK1 cells. Uncoupling and loss of vasopressin receptors. 296 82

Previous studies have provided pharmacologic evidence that T lymphocyte function may be regulated in part by the intracellular production of various arachidonic acid (AA) metabolites in response to cellular stimulation. However, the specific AA metabolic capabilities of homogeneous T cell populations have not been clearly defined. In the present studies, we have employed an accessory cell-free T cell line, HT-2, as a model system for the examination of stimulus-induced eicosanoid biosynthesis in T lymphocytes. HT-2 cells were biosynthetically labeled with [3H]-AA and challenged briefly with various agents that stimulate the hydrolytic release of AA from cellular phospholipids. The bee venom peptide melittin stimulated a profound AA release response in the cells and the concomitant synthesis of both cyclooxygenase (PGF2 alpha, PGE2 and PGD2) and lipoxygenase (5-,12-,15-HETE and possibly 5-,12-diHETE) metabolites of AA. The formation of PGs was blocked by 5 microM indomethacin, demonstrating that this cell line contains cyclooxygenase activity functionally similar to that described in macrophages and other cell types. The high activity of melittin in this system was shown to result largely from a synergy between the peptide itself and a persistent bee venom phospholipase A2 contaminant. However, experiments with melittin freed of detectable phospholipase A2 activity by heating, and with synthetic homopolymers of (L)-lysine and (L)-arginine demonstrated that HT-2 cells contain sufficient endogenous, stimulus-responsive phospholipase A2 to provide both the cyclooxygenase and lipoxygenase pathways of AA metabolism ith substrate. In contrast, Ca++ ionophores, which are known to stimulate AA release and metabolism in certain cell types, stimulated only AA release but no detectable eicosanoid biosynthesis in HT-2 cells. Experiments with exogenous bacterial phospholipase C suggested that this cell line can also generate free AA for eicosanoid biosynthesis from membrane-derived 1,2-diacylglycerol. These results indicate that multiple intracellular pathways of AA metabolism are present HT-2 cells, and that the stimulus-induced release of AA and the production of eicosanoid second messengers may result from activation of either phospholipase A2 or phospholipase C.
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PMID:Stimulation of arachidonic acid release and eicosanoid biosynthesis in an interleukin 2-dependent T cell line. 308 27

We labeled cobra-alpha-toxin (Naja naja siamensis 3) with near stoichiometric quantities of fluorescein isothiocyanate. To reduce labeling of the hyperreactive N epsilon-lysine 23, the alpha-toxin was modified reversibly with citraconic anhydride before fluorescein labeling. The citraconic anhydride was later removed with strong acid, and four of the six possible monofluorescein alpha-toxin derivatives were isolated by isoelectric focusing on an immobilized pH gradient. Thermolysin digestion and subsequent high pressure liquid chromatography of the peptides yielded one dominant fluorescent peak from three of the isolated monofluorescein derivatives. Sequence analyses of these three fluorescent peaks indicated monofluorescein labeling at Lys-69, Lys-35, and Lys-49. Since one derivative (not identified by sequence analysis) displayed essentially identical chromatographic, spectroscopic, and binding properties as our previously identified monofluorescein-Lys-23 toxin (Johnson, D. A., and Taylor, P. (1982) J. Biol. Chem. 257, 5632-5636), we identified the site of labeling of this fourth derivative to be Lys-23. While only small differences were observed in the extinction maxima and molar extinction coefficients, the quantum yields of the isolated derivatives varied markedly and ranged between 0.18 and 0.41. Binding of monofluorescein-Lys-69, -Lys-35, -Lys-49, and -Lys-23 derivatives to the membrane-associated acetylcholine receptor from Torpedo californica was associated with -39, -26, -9, and +96% changes in fluorescence emission intensity, respectively. Based on analyses of the kinetics of fluorescence changes associated with receptor binding, the association and dissociation rate constants were measured. Relative to native cobra alpha-toxin, monofluorescein conjugation reduced the bimolecular association rate constants for binding to the receptor 13-33-fold. The dissociation rate binding rate constants were less affected and were reduced 0-5-fold.
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PMID:Purification and characterization of four monofluorescein cobra alpha-toxin derivatives. 312 69

Orthorhombic crystals (space group P212121, a = 45.94 A, b = 40.68 A, c = 29.93 A) of the potent scorpion alpha-toxin II from Androctonus australis Hector were grown using sterile techniques. The structure was solved by a combination of heavy-atom and model phasing. Subsequently, it was refined at 1.8 A resolution by a fast-Fourier restrained least-squares procedure. The crystallographic R factor is 0.152 for data with 7.0 A greater than d greater than 1.8 A and F greater than 2.5 sigma (F) and 0.177 when all data are considered. Eighty-nine solvent molecules have been incorporated into the model. The dense core formed by the alpha-helical and antiparallel beta-sheet moieties and three of the four disulfide bridges is similar in variant 3, a toxin purified from the North American scorpion Centruroides sculpturatus, and in toxin II. However, the two molecules differ markedly in the orientation of loops protruding from the core. Toxin II seems to contain several highly ordered solvent molecules. Eight of them occupy a cavity consisting of the C-terminal region and a loop found only in scorpion alpha-toxins. The highly reactive and pharmacologically important Lys-58 is found at one of the extremes of this cavity, where it establishes a series of hydrogen bonds with protein and solvent atoms. The reactivities of the five lysine residues of toxin II are highly correlated with the formation of hydrogen bonds, hydrophobic interactions, and salt links.
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PMID:Orthorhombic crystals and three-dimensional structure of the potent toxin II from the scorpion Androctonus australis Hector. 317 45

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.
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PMID:Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid. 333 45

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

To obtain information on the disposition of alpha-toxin when bound to the acetylcholine receptor (AChR), we evaluated the accessibility of solutes to fluorescein isothiocyanate (FITC) conjugated to alpha-toxin (siamensis 3) at lysine 23 (FITC-toxin) by measuring the rate constants for iodide quenching of the fluorescence of fluorescein free in solution and FITC-toxin free in solution and bound to AChR. Relative to the free fluorescein, we observed a 55% reduction in the quenching rate constant for the unbound FITC-toxin and 80% reduction for the AChR-bound FITC-toxin. It is tempting to interpret a decrease in the quenching rate constant as due to an increase in the masking of the labeling fluorophore, which in our case would then be indicative of masking of fluorescein conjugated to the free toxin and masking of FITC-toxin, in the region of lysine 23, when bound to AChR. However, elementary considerations indicate that the quenching rate depends not only on geometrical masking factors but also on the translational and rotational mobilities of the labeled molecules as well as orientational constraints. To evaluate these effects we have established quantitative relations between the rate of fluorescence quenching, the degree of masking of fluorophore, translational and rotational rates, and orientational constraints of the labeled macromolecules, using recent formulations for the rate of reaction between asymmetric molecules (Shoup et al., 1981, Biophys. J., 36:619-714). These relations predict that the decrease in quenching constant observed for the labeled FITC-toxin as well as the AChR-bound FITC-toxin is largely due to differences in translational and rotational rates and orientational constraints and not to significant increases in geometrical masking. Our theoretical formulation shows that the quenching rate can be decreased by a factor of 2-5 merely by immobilizing a fluorophore on the surface of a large protein without any significant increase in geometrical masking.
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PMID:Solute accessibility to N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin bound to the acetylcholine receptor. A consideration of the effect of rotational diffusion and orientation constraints on fluorescence quenching. 393 57

The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane. A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.
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PMID:Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode, Ascaris suum. Nature of the electronegative charge. 615 29


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