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)

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.
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PMID:Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein. 302 97

Inhibitors of pancreatic islet lipoxygenase (LPX) impair nutrient-induced insulin (I) release. To define the mechanism of action of these inhibitors, studies were carried out at subthreshold glucose concentrations (0-1.7 mM) in order to minimize any effects of LPX blockade on the potentiating effect of extracellular fuels. Barium chloride (Ba2+; 2 mM) increased 45Ca2+ release from prelabeled islets in Ca2+-free medium and, thus, is a model for the mobilization of intracellular Ca2+ stores. Inhibition of LPX (using nordihydroguaiaretic acid, BW755c [3-amino-1-(trifluomethyl-phenyl)2-pyrazoline] or butylated hydroxytoluene) did not have any consistent effect on the influx of Ba2+ (as assessed by 133Ba uptake) or on the consequent release of cellular Ca2+ stores; however, each LPX inhibitor vitiated Ba2+-induced I release. The LPX inhibitors were not merely acting as nonspecific antioxidants, since two inhibitors which do not act by scavenging hydroperoxides (5,8,11,14-eicosatetraynoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid) also impeded the effect of Ba2+ on I secretion; furthermore, a series of hydroxyl radical scavengers, reducing agents, or agents that block nonenzymatic and/or NADPH-activated lipid peroxidation did not inhibit I secretion. LPX inhibitors also blocked the residual I response to 16.7 mM glucose in Ca2+-free medium. Additionally, they reduced secretion induced by 46 mM K+ or 1 mM isobutylmethylxanthine (provided in the presence of extracellular Ca2+), without inhibiting K+- or isobutylmethylxanthine-induced Ca2+ fluxes. Stimuli sensitive to LPX blockade were also antagonized by antimycin A (an inhibitor of energy flux) or TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; which appeared to deplete critical intracellular Ca2+ stores]. In contrast, the effects of exogenous phospholipase C (and several other Ca2+-dependent membrane-active agonists) were resistant to the LPX inhibitors, TMB-8, and antimycin A; thus, LPX inhibitors are not nonspecific global poisons of all Ca2+-dependent exocytotic hormone release. We conclude that LPX (or a very similar enzyme) may modulate the effects (or redistribution) of an ATP-dependent trigger pool of Ca2+ at a site distal to and independent of its mobilization by primary islet agonists. LPX inhibitors also blocked secretion induced by 12-O-tetradecanoyl-phorbol-13-acetate; this effect may reflect an effect of LPX on the activation of protein kinase C or a modulation of its synergism with the same trigger Ca2+ pool(s).
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PMID:Lipoxygenase inhibitors reduce insulin secretion without impairing calcium mobilization. 310 21

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.
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PMID:Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides. 337 61

Carbon tetrachloride (CCl4) biotransformation and covalent binding was measured in 1000g liver fractions by determining the amount of 14CCl4 metabolites covalently bound to proteins and lipids at various (5-60 min) incubation times. Reactive intermediate binding to proteins and phospholipids peaked at 20 min, whereas CCl4 metabolites associated with neutral lipids (primarily diacylglycerol) were initially low (0-15 min) and then gradually increased from 20 to 60 min. The rise in labeled diacylglycerol was associated with a decrease in phospholipids containing covalently bound CCl4 metabolites, since CCl4 bioactivation increased phospholipase C (PLC) activity three- to fourfold. The major rise in PLC activity occurred after the plateau of CCl4 metabolite binding to cellular phospholipids. In contrast, when CCl4 bioactivation is absent, 0.5 mM CCl4 has little effect on PLC activity. At CCl4 concentrations of 1 mM and greater, the NADPH-dependent activation of PLC by CCl4 is reduced because CCl4 biotransformation is inhibited. Nevertheless, PLC is still activated by CCl4; however, PLC activation by high doses of CCl4 occurs by bioactivation-independent mechanisms. Therefore, there are two components of CCl4-induced PLC activation: one which is dependent on CCl4 biotransformation and one which is not. Under both conditions (+/- biotransformation), the activation of PLC may be a key event in CCl4 hepatotoxicity since PLC disrupts the functional and structural integrity of membranes by degrading membrane phospholipids.
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PMID:The influence of CCl4 biotransformation on the activation of rat liver phospholipase C in vitro. 342 Jun 12

The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca2+]i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca2+]i, it restored the burst response to agonists in Ca(2+)-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca(2+)-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G(i)-proteins, but that in the presence of PMA portions of the agonist response could be recovered.
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PMID:Activation of the respiratory burst in eosinophil leucocytes--a transduction sequence decoupled from cytosolic Ca2+ rise. 770 83

Thioredoxin (Trx) catalyzes thiol-disulfide oxidoreductions. We and others recently showed that human Trx could function as an autocrine growth factor for human lymphoid cells immortalized by the human T-lymphotrophic virus type I or the Epstein-Barr virus. Here we report that reduced Trx from Escherichia coli generated by NADPH and thioredoxin reductase increases the proliferation of an Epstein-barr virus(+)-B cell line 1G8, which constitutively produces low amounts of human Trx. This proliferative effect involved the activation of protein kinase C through its translocation to the membrane. Staurosporin and calphostin C, two inhibitors of protein kinase C, but not of H8, a protein kinase A inhibitor, were able to block Trx-dependent proliferation. The addition of Trx to 1G8 cells resulted in the formation of inositol 1,4,5-triphosphate and sn-1,2-diacylglycerol by a phosphoinositide-specific phospholipase C, as well as increased free calcium concentration. Diacylglycerol showed a biphasic increase; the first phase, corresponding to an early peak (30 s) of inositol 1,4,5-triphosphate and a second larger, prolonged phase. The second phase was inhibited by propranolol, a specific inhibitor of phosphohydrolase, indicating that it is most likely derived from phosphatidylcholine hydrolysis by the sequential action of phospholipase D and phosphatidic acid phosphohydrolase. Our data suggest that enhanced phosphoinositide-specific phospholipase C activity induced by the dithiol form of Trx in 1G8 cells is associated to protein kinase C activation, and thus plays a role in the permanent growth of Epstein-Barr virus-infected B cells.
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PMID:Thioredoxin increases the proliferation of human B-cell lines through a protein kinase C-dependent mechanism. 796 46

The respiratory burst oxidase from human neutrophils is a membrane-associated enzyme that catalyzes the reduction of oxygen to O2- at the expense of NADPH. The oxidase is dormant in resting neutrophils, but comes to life when the cells are exposed to certain activating agents. Activation requires GTP or a GTP analog and is associated with the transfer of cytosolic oxidase polypeptides to the plasma membrane. Treatment of resting neutrophil membranes with phospholipase C caused an eightfold rise in their diacylglycerol content, increased the sodium dodecyl sulfate-mediated transfer of cytosolic polypeptides to the membrane, and enhanced O2- production by the membranes after treatment with cytosol and sodium dodecyl sulfate. Use of phospholipase C-treated membranes in the cell-free system caused only a minor change in the Km and Vm for NADPH as compared with the same system containing untreated membranes, but caused the Km for the nonhydrolyzable GTP analog GTP gamma S to fall from 200 to 34 nM. Similar kinetic changes were observed with membranes treated with dioctanoylglycerol. These findings are consistent with the idea that the activity of a G protein can be regulated by diacylglycerol.
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PMID:Effects of diacylglycerol on the activation and kinetics of the respiratory burst oxidase in a cell-free system from human neutrophils: evidence that diacylglycerol may regulate nucleotide uptake by a GTP-binding protein. 821 91

1. The ability of acetylshikonin to inhibit the respiratory burst in rat neutrophils was characterized and the underlying mechanism of action was also assessed in the present study. 2. Acetylshikonin caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.48 +/- 0.03 and 0.39 +/- 0.03 microM, respectively. Acetylshikonin also inhibited the O2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3. Acetylshikonin did not scavenge the generated O2.- in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O2.- generation in these cell-free oxygen radical generating systems. 4. Acetylshikonin inhibited the formation of inositol trisphosphate (IP3) (39.0 +/- 7.8% inhibition at 10 microM, P < 0.05) in neutrophils in response to fMLP. 5. Both the neutrophil cytosolic protein kinase C (PKC) activity and the PMA-induced PKC associated with the membrane were unaffected by acetylshikonin. 6. Acetylshikonin did not affect the porcine heart protein kinase A (PKA) activity. Upon exposure to acetylshikonin, the cellular cyclic AMP level was decreased in neutrophils in response to fMLP. 7. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP/CB were inhibited by acetylshikonin (60.1 +/- 7.3 and 63.2 +/- 10.5% inhibition, respectively, at 10 microM, both P < 0.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 microM). 8. In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O2.- generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47phox in PMA-activated neutrophils (about 60% inhibition at 1 microM). 9. Collectively, these results suggest that the attenuation of protein tyrosine phosphorylation and a failure in the assembly of a functional NADPH oxidase complex probably contribute predominantly to the inhibition of respiratory burst in neutrophils by acetylshikonin. In contrast, the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways play only a minor role in this respect.
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PMID:Investigation of the inhibition by acetylshikonin of the respiratory burst in rat neutrophils. 917 81

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O2.-) generation and O2 consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O2.- generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca2/]i and the formation of inositol trisphosphate (IP3) were significantly inhibited by norathyriol (30 microM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 microM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O2.-, however, the inhibition of O2.- generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.
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PMID:Examination of the inhibitory effect of norathyriol in formylmethionyl-leucyl-phenylalanine-induced respiratory burst in rat neutrophils. 935 47

This paper is addressed to study how PKC-mediated effects and phosphatidic acid interact together in activation of NADPH-oxidase in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) stimulated neutrophils as detected by luminol chemiluminescence. The early luminescence response in fMet-Leu-Phe-stimulated cells (up to 5 min after stimulation) depends mainly on reactive oxygen species generated extracellularly, whereas all later events are caused by oxidation of luminol inside the cells. The two protein phosphatase inhibitors, okadaic acid and calyculin A, dramatically increased the late luminescence of cells. This enhancement was totally inhibited by the phospholipase D modulator butanol, while the protein kinase C (PKC) inhibitor bisindolylmaleimide I was insensitive. The early luminescence response of the cells was slightly inhibited by both protein phosphatase inhibitors and depended on protein kinase C as well as on phospholipase D activities. Propranolol, an inhibitor of phosphatidate phosphohydrolase, enhanced all parts of luminescence response of fMet-Leu-Phe-stimulated neutrophils at concentrations up to 2.5 x 10(-5) mol/L. While the late luminescence response of propranolol-treated cells was not inhibited by the PKC inhibitor bisindolylmaleimide I, the first response depended on protein kinase C. The inhibitor of diacylglycerol kinase R59949 enhanced the luminescence signal only during the first 4 min in fMet-Leu-Phe-stimulated cells. Only diacylglycerols derived from phospholipase C, such as 1-stearoyl-2-arachidonoyl-sn-glycerol, were able to initiate an oxidative burst in cells. Saturated diacylglycerols (e.g. 1,2-dipalmitoyl-sn-glycerol or 1,2-distearoyl-sn-glycerol) did not yield any luminol chemiluminescence, although they were incorporated into the plasma membrane, as evidenced by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Our results demonstrate that phosphatidic acid produced by phospholipase D is responsible for NADPH-oxidase activity in fMet-Leu-Phe-stimulated neutrophils over the entire measuring time, whereas PKC-mediated processes are only involved during the first 5 min.
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PMID:Modulation of luminol chemiluminescence of fMet-Leu-Phe-stimulated neutrophils by affecting dephosphorylation and the metabolism of phosphatidic acid. 1042 73


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