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)

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.
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PMID:Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver. 1 Aug 91

[3-H]Epinephrine binding to isolated purified rat liver plasma membranes is a reversible process. An initial peak in binding occurs at about 15 min and a plateau occurs by 50 min. Optimal binding occurred at a membrane protein concentration of 125mug. Rat liver plasma membranes stored at-70 degrees C up to 4 weeks showed no difference in epinephrine binding capacity as compared to control fresh membranes. Epinephrine binding to liver plasma membranes was decreased by 79% by phospholipase A2 (phosphatide acylhydrolase EC 3.1.1.4), 81% by phospholipase C (phosphatidylcholine choline phosphohydrolase EC 3.1.4.3) and 59% by phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4). Trypsin and pronase digestion of the membrane decreased epinephrine binding by 97 and 47% respectively. In the presence of 10-3M Mg-2+ ions, increasing concentrations of QTP decreased epinephrine binding to liver plasma membranes. A maximal effect was demonstrated with 10-5M GTP, representing an inhibition of 52% of the control. In a Mg-2+ -free system, epinephrine binding was unaffected by GTP. However, in a Mg-2+ -free system, increasing concentrations of ATP cause increasing inhibition of hormone binding. ATP at 10-3 M reduced epinephrine binding to 28% of the control. GRP (10-5 M) was shown to inhibit epinephrine uptake rather than epinephrine release from the membrane. [3-H]Epinephrine binding to isolated rat epididymal fat cells shows an initial peak within 5 min followed by a gradual rise which plateaus after 60 min. Epinephrine binding increased nearly linearly with increasing fat cell protein concentration (40-200 mug protein). GTP (10-5 M) and ATP (10-4 M) decreased epinephrine binding to rat epididymal fat cells by 41%. Nearly complete inhibition of binding was demonstrated with 10-2-10-3M ATP. Epinephrine analogs that contain two hydroxyl groups in the 3 and 4 position on the benzene ring act as inhibitors of [3-H]epinephrine binding to rat adipocytes. Alteration of the epinephrine side chain has relatively little influence on binding. Analogs in which one of the ring hydroxyl groups is missing or methylated are poor inhibitors of [3-H]epinephrine binding. Alpha-(phentolamine and phenoxybenzamine) and beta-(propranolol and dichorisoproterenol) adrenergic blocking agents were tested with respect to their ability to influence [3-H]epinephrine binding and their influence on epinephrine-stimulated lipolysis. Only dichloroisoproterenol significantly inhibited epinephrine binding (by 25%). The two beta-adrenergic blocking agents caused an inhibition of epinephrine-stimulated glycerol release, with propranolol being most effective. Phentolamine and phenoxybenzamine had no significant effect on the epinephrine stimulation of glycerol release by fat cells.
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PMID:Hormone action at the membrane level. IV. Epinephrine binding to rat liver plasma membranes and rat epididymal fat cells. 16 9

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.
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PMID:Activation of guanylate cyclase from rat liver and other tissues by sodium azide. 24 Aug 48

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.
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PMID:Effects of phospholipase A2 and filipin on the activation of adenylate cyclase. 42 Aug 40

Previous studies have demonstrated that although both mammalian and avian erythrocytes express an inducible inositol bisphosphate-specific phospholipase C, only the latter possess the guanine nucleotide-binding protein (Gp) that regulates this activity. In confirmation of previous reports, turkey erythrocyte plasma membranes responded to guanosine 5'-0-(3-thio)triphosphate (GTP-gamma-S) and fluoroaluminates with hydrolysis of phosphoinositides, release of inositol phosphates, and generation of diacylglycerol, whereas human erythrocyte plasma membranes exhibited no such changes when incubated with known activators of guanine nucleotide regulatory proteins. We next contrasted responses of intact turkey and human erythrocytes to fluoroaluminates to develop a model to investigate the cellular effects of Gp activation. When turkey erythrocytes were exposed to fluoroaluminates, cellular levels of diacylglycerol and phosphatidic acid rapidly increased as phosphoinositides were hydrolyzed. The alterations in the lipid composition of turkey erythrocytes effected by fluoroaluminates were remarkable; phosphatidic acid levels increased over 30-fold, whereas levels of polyphosphoinositides were decreased to less than 10% of those present before stimulation. In contrast, fluoroaluminates caused only minor alterations in the diacylglycerol and phospholipid content of intact human erythrocytes. To define the role of inositol-specific phospholipase C activation in the transmembrane conveyance of extracellular Ca++, we compared the influx of extracellular Ca++ in human and turkey erythrocytes exposed to fluoroaluminates. Fluoroaluminates initiated a sustained influx of extracellular 45Ca++ into turkey, but not human, erythrocytes. These results provide strong support for the hypothesis that Gp activation results in an influx of calcium into stimulated cells. Moreover, the data demonstrate that comparison of responses of human and turkey erythrocytes to fluoroaluminates provides a well-defined method for investigating the mechanisms and consequences of Gp activation in intact cells.
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PMID:Gp-regulated phosphoinositide hydrolysis in turkey and human erythrocytes exposed to fluoride ion: relationship to calcium influx. 130 78

The effect of quisqualate, an excitatory amino acid agonist, on the breakdown of exogenously added phosphatidylinositol was investigated in a membrane preparation from the cerebellum of young rats. Quisqualate stimulated phospholipase C activity in a dose-dependent manner in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Half-maximal activation of the quisqualate response required 0.15 microM GTP gamma S and was optimal at a free Ca2+ concentration of 300 nM. Phosphoinositide breakdown was also stimulated by quisqualate using either exogenous phosphatidylinositides 4,5-bisphosphate or endogenous labeled phosphoinositides as the substrate for phospholipase C in cerebellar membranes. In the presence of guanine nucleotides, other excitatory amino acid agonists, such as L-glutamate, trans-D,L-1-aminocyclopentyl-1,3-dicarboxylic acid, and ibotenate, but not N-methyl-D-aspartate, stimulated phosphatidylinositol breakdown. However, quisqualate displayed the highest response among these excitatory amino acid agonists. These data indicate that there is a direct activation of phosphoinositide-specific phospholipase C by excitatory amino acids through a process dependent on the presence of guanine nucleotides.
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PMID:Quisqualate-stimulated phosphatidylinositol breakdown in rat cerebellar membranes. 130 70

Signal transduction pathways may have important regulatory roles in cellular events in the human parasite Schistosoma mansoni. The presence of the phosphoinositide response in S. mansoni was examined by radiolabeling intact worms with 20 muCi of [3H]myoinositol for 24 hr and stimulating parasites with 25 mM NaF and 10 microM AlCl3 in the presence of 10 mM LiCl. Total inositol phosphates were increased within 2 min and maximal accumulation was achieved after 30 min. Similar results were seen with the non-hydrolyzable GTP analogues GTP gamma S and GppNHp while only minimal changes were detected with GMP. Neomycin inhibited NaF-induced inositol phosphate production. NaF stimulated a significant 3.6-fold increase of inositol phosphates in females compared to males. These data suggest that stimulation of guanine nucleotide-binding regulatory proteins activates phospholipase C resulting in production of inositol phosphates in S. mansoni.
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PMID:Schistosoma mansoni: characterization of phosphoinositide response. 130 1

The effect of the stable cAMP analogue 8-Br-cAMP on leukotriene D4 (LTD4)-, 5'-N-ethyl-carboxamidoadenosine (NECA)-, antigen- and Ca2+ ionophore-induced inositol phosphate (IP) production was studied in RBL-1 cells. The cAMP analogue significantly inhibited LTD4- and antigen induced-IP production, thus supporting the hypothesis of a negative interaction between cAMP and phosphoinositide breakdown in blood cells. Ionophore-induced IP release, which was blocked by a 5-lipoxygenase inhibitor and by a LT-receptor antagonist, and therefore is probably mediated by LTs, was also inhibited by 8-Br-cAMP. NECA-induced IP release was not significantly inhibited by the cyclic nucleotide, thus showing that the effect described herein is not a general action on receptor-activated phospholipase C. 8-Br-cAMP did, however, inhibit GTP gamma S-induced IP release in permeabilised RBL-1 cells, thus suggesting that the inhibition does not occur at the receptor level but might be due, at least in part, to an effect on some receptor-coupled G proteins.
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PMID:The effect of a cAMP analogue on Ca2+ ionophore-, antigen- and agonist-induced inositol phosphate release in rat basophilic leukaemia (RBL-1) cells. 131 54

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.
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PMID:Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion. 131 May 99

Platelet-activating factor (PAF) is an unusually potent phospholipid known to be produced by neuronal cells and to modulate cerebral blood flow and metabolism. In previous studies with NCB-20 cells, we reported that PAF induced a significant mobilization of intracellular free Ca2+ ([Ca2+]i), which was inhibited by PAF antagonists. The increase was the result of release from intracellular stores and influx from extracellular sources. The present study was designed to characterize further PAF receptor-mediated cellular signal-transduction mechanisms in myo-[3H]inositol-labeled cells. PAF induced a concentration-dependent increase in phosphatidylinositol (Pl) metabolism, with EC50 values of 1.96 +/- 0.62 nM and 1.12 +/- 0.50 nM for inositol trisphosphate (IP3) and inositol monophosphate (IP1) formation, respectively (four experiments). The maximal production of IP3 and IP1 induced by 50 nM PAF was 254 +/- 34% and 178 +/- 25% over the basal, respectively (four experiments). PAF-induced Pl metabolism was concentration-dependently inhibited by the PAF antagonist BN50739, with an IC50 value of 6.48 +/- 0.52 nM (four experiments). The protein kinase C (PKC) activator phorbol 12,13-dibutyrate concentration-dependently inhibited PAF-induced Pl metabolism and [Ca2+]i mobilization in NCB-20 cells, of NCB-20 cells with pertussis toxin (PTX) resulted in a concentration-dependent inhibition of PAF-induced IP3 production and intracellular Ca2+ release, with a maximal reduction of 66.9 +/- 3.5% and 63 +/- 6.1%, respectively, at 300 ng/ml PTX. PTX in the presence of [32P]NAD specifically [32P]ADP-ribosylated a 38-kDa protein in membranes prepared from NCB-20 cells. Pretreatment of the cells with PTX resulted in a concentration-dependent inhibition of subsequent 32P-labeling of the toxin substrate in the membranes and correlated with the uncoupling of PAF-induced IP3 formation. PAF (0.01-10 nM) elicited a concentration-related stimulation in guanosine 5'-O-(3-[35S]) triphosphate ([35S]GTP gamma S) binding to G alpha i(1,2) proteins, which was inhibited by the PAF antagonist BN50739. PAF at 10 nM also increased [35S]GTP gamma S binding to G alpha s and G alpha o. PAF-evoked activation of G alpha i(1,2) and G alpha o was reduced by preincubation with PTX. Our results reveal that neuronal cells possess PAF receptors linked through guanine nucleotide-binding proteins to phospholipase C and receptor-operated Ca2+ channels that are regulated by PKC. Both PTX-sensitive and -insensitive guanine nucleotide-binding proteins appear to couple the PAF receptor to activation of phospholipase C and the increase in [Ca2+]i. These results contribute to the further understanding of the mechanisms behind PAF actions on neuronal cells.
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PMID:Platelet-activating factor stimulates phosphoinositide turnover in neurohybrid NCB-20 cells: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins and inhibition by protein kinase C. 131 8


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