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)

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.
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PMID:Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity. 254 69

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10(-4) M) or guanosine 5'-0-(3-thiotriphosphate, GTP gamma S, 10(-5) M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTP gamma S produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H]IP3, respectively. The stimulatory effects of GTP and GTP gamma S were dose dependent (GTP ED50 = 3.9 x 10(-6) M; GTP gamma S ED50 = 2.5 x 10(-7) M) and progressive over 10 minutes and required the presence of Mg2+.GTP (10(-4) M) and GTP gamma S (10(-5) M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDP beta S) alone did not alter [3H]IP2 or [3H]IP3 production but at 10(-4) M blocks the stimulatory effects of GTP and GTP gamma S. NaF (3 x 10(-2)M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes. 255 86

Phosphatidylinositol 3-phosphate (PtdIns(3)P), a recently described phospholipid, has been linked to polyoma virus-induced cellular transformation and platelet-derived growth factor-mediated mitogenesis. PtdIns(3)P, in contrast to phosphatidylinositol, phosphatidylinositol 4-phosphate (PtdIns(4)P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), is resistant to hydrolysis by bovine brain phospholipase C gamma. We present here the identification of a phosphomonoesterase activity from the soluble fraction of NIH 3T3 cells which removes the phosphate from the D-3 position of PtdIns(3)P. This enzyme is specific as it has little or no activity on the monoester phosphates of PtdIns(4)P, PtdIns(4,5)P2, or inositol 1,3-bisphosphate and is tentatively designated phosphatidylinositol 3-phosphatase (PtdIns 3-phosphatase). The enzyme does not require added metal ions for activity and is maximally active in the presence of EDTA. It is inhibited by Ca2+, Mg2+, Zn2+, and the phosphatase inhibitor VO4(3-). In addition, there is no phospholipase C activity toward PtdIns(3)P in the soluble fraction of NIH 3T3 cells. In view of the absence of a phospholipase C activity that hydrolyzes PtdIns(3)P, we propose that PtdIns(3)P is not a precursor for a soluble inositol phosphate messenger but that it instead may act directly to control certain cellular processes or as a precursor for other phosphatidylinositols. PtdIns 3-phosphatase may thus terminate a metabolic signal or regulate precursor levels for other phosphatidylinositols that are phosphorylated in the D-3 position.
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PMID:The discovery of a 3-phosphomonoesterase that hydrolyzes phosphatidylinositol 3-phosphate in NIH 3T3 cells. 255 36

To evaluate the regulation and effects of pancreatic islet lipoxygenase, adult rat islets were permeabilized, using digitonin or staphylococcal alpha-toxin, and then were studied in a medium simulating an intracellular milieu at fixed ambient concentrations of Ca2+. Permeabilized islets retained 12-lipoxygenase activity, as indicated by conversion of tritiated arachidonic acid to a predominant peak of [3H]12-hydroxyeicosatetraenoic acid (12-HETE); this activity was inhibited (89-98%) by the lipoxygenase blockers nordihydroguaiaretic acid (35 microM), BW755c (250 microM) or ETYA (35 microM). Lesser amounts of compounds coeluting with 15- and 11-HETE (but little or no 5-HETE) were formed; however, 11-HETE (and possibly some 15-HETE) was probably synthesized (at least in part) via cyclooxygenase, as suggested by the partial synthesis blockade induced by 50 microM ibuprofen. The production of 12-HETE did not require the presence of Ca2+, Mg2+ or ATP; it also was not stimulated by addition of cyclic AMP, a phorbol ester, or calmodulin. However, it was augmented modestly by provision of a basal cytosolic free Ca2+ concentration of 60-80 nM, with no further increase at physiologically elevated levels of 260-530 nM. Elevations in cytosolic free Ca2+ concentrations induced insulin release which was inhibited by cooling, epinephrine or protein kinase inhibitors and, therefore, was exocytotic in nature. Lipoxygenase inhibitors blocked this insulinotropic effect of calcium at submaximal or saturating Ca2+ concentrations (with or without its potentiation by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C) by 53-82%. However, they did not reduce the Ca2+-independent secretory effects (at subnanomolar Ca2+ concentrations) of the phorbol ester alone. Similar results were seen using dibutyryl cyclic AMP to activate protein kinase A. The alpha 2-adrenergic agonists epinephrine or clonidine inhibited Ca2+-, TPA- or cyclic AMP-induced insulin release without reducing HETE formation. We conclude that (1) islet lipoxygenase is constitutively expressed and is not physiologically regulated by alpha 2-adrenergic agonism, Ca2+ or protein kinases; (2) lipoxygenase modulates insulin release; HETE production is not merely an epiphenomenon reflecting the activation (or inhibition) of exocytotic secretion; (3) islet lipoxygenase inhibitors reduce insulin secretion, at least in part, by blocking the direct effects of Ca2+ on exocytosis and/or its synergism with Ca2+-binding proteins such as protein kinase C; and (4) these same inhibitors do not directly poison protein kinase C or A, or the exocytotic apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blockade by lipoxygenase inhibitors of Ca2+-dependent insulin secretion from permeabilized rat islets. A molecular mechanism distinct from that of alpha 2-adrenergic agonists. 256 95

Excitatory amino acids (EAA) are known to induce an increase in the breakdown of polyphosphoinositides (PI) in brain slices and in dispersed cultures of neurons. We have now used astroglia cultured from newborn rat cerebra to demonstrate that glutamate provokes, in [3H]inositol-labeled cells, an accumulation of inositol phosphates in a time- and concentration-dependent manner. The ED50 value for glutamate was 40 microM. Quisqualate, ibotenate, and kainate were also active, with their relative potencies in the order of quisqualate greater than ibotenate much greater than kainate. No effect was detected with N-methyl-D-aspartate and quinolinic acid in the absence of Mg2+. The nonselective glutamate receptor antagonist gamma-D-glutamylglycine fully inhibited glutamate agonist-induced PI breakdown. A brief pretreatment of the astroglial cells with phorbol esters negated these effects of EAA receptor agonists, suggesting a feedback role for protein kinase C in phospholipase C action. Glutamate also elevated cytosolic free Ca2+ in Fura-2-loaded astroglial cells, as assessed by digital fluorescence imaging microscopy. Since a close metabolic partnership is known to exist between neurons and glia, these findings may have important functional consequences for neural cells in vivo.
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PMID:Activation of polyphosphoinositide metabolism as a signal-transducing system coupled to excitatory amino acid receptors in astroglial cells. 256 42

In human skin fibroblasts, low concentrations of extracellular ATP stimulated 45Ca2+ efflux from a slow-turnover intracellular pool, accompanied by inositol phosphate generation. These effects of ATP were not due to a generalized increase in plasma-membrane permeability. The EC50 (concn. giving 50% stimulation) for ATP was dependent on Ca2+ and Mg2+ concentrations in a manner which indicates that a form of ATP uncomplexed with bivalent cations is the active species. The rank order of potency of nucleotides was: ATP = UTP greater than adenosine 5'-[gamma-thio]triphosphate greater than ITP greater than ADP greater than UDP greater than other nucleoside triphosphates. Adenosine 5'-[alpha beta-methylene]triphosphate, adenosine 5'-[beta gamma-methylene]triphosphate and 2-methylthio-ATP were inactive. Thus the nucleotide specificity of this receptor is different from that of previously characterized P2 purinoceptors. Nucleotide-stimulated 45Ca2+ mobilization and inositol phosphate production were markedly inhibited by phorbol ester, and partially inhibited by pertussis-toxin pretreatment. These findings suggest that the coupling of nucleotide receptor to phospholipase C is mediated both by a pertussis-toxin-sensitive G-protein and by a pertussis-toxin-insensitive mechanism.
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PMID:Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts. 259 9

The role of Mg2+ during the final steps of exocytosis was investigated using rat pheochromocytoma cells (PC12) permeabilized with bacterial pore forming toxins. Concentrations of free Mg2+ between 0.2 and 2 mM slightly lowered the basal but greatly enhanced the [3H]dopamine release elicited by 8 microM free Ca2+. Maximal effects were obtained at approximately 1 mM free Mg2+. At higher concentrations Mg2+ was less potent. Similar effects of Mg2+ were obtained in cells permeabilized either for small molecules (by alpha-toxin) or for large ones (by streptolysin O). It is concluded that millimolar concentrations of cytoplasmic Mg2+ play an important role in Ca2+ triggered exocytosis.
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PMID:Millimolar concentrations of free magnesium enhance exocytosis from permeabilized rat pheochromocytoma (PC 12) cells. 260 84

In cerebral-cortical membranes, hydrolysis-resistant guanine nucleotides exert a dual regulatory effect on phospholipase C activity. Nanomolar concentrations of guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited basal phospholipase C activity, with a maximum inhibition of 30% at 10 nM. Increasing the concentration of p[NH]ppG or GTP[S] to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of phospholipase C activity. These inhibitory effects were blocked by 100 microM-guanosine 5'-[beta-thio]diphosphate. GTP was relatively ineffective in producing either stimulation or inhibition of phospholipase C activity. Similarly, ATP, adenosine 5'-[beta gamma-imido]triphosphate and GDP were also ineffective. Expression of the dual effects of guanine nucleotides was affected by the Mg2+ concentration. At 0.3 mM-Mg2+, both the inhibitory and the stimulatory components of p[NH]ppG action were evident. At 2.5 mM-Mg2+, only p[NH]ppG stimulation was observed. Pertussis-toxin treatment blocked the p[NH]ppG-mediated inhibition of phospholipase C activity. These results demonstrate that non-hydrolysable guanine nucleotides exert both a stimulatory and an inhibitory effect on membrane phospholipase C activity. These effects may be mediated through distinct GTP-binding proteins.
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PMID:Guanine nucleotides mediate stimulatory and inhibitory effects on cerebral-cortical membrane phospholipase C activity. 267 14

We have previously shown that interleukin-1 (IL-1) rapidly stimulates the hydrolysis of phosphatidylcholine in the human T lymphocyte cell line, Jurkat (Rosoff, et al., Cell 54: 73-81, 1988). This was apparently mediated by a phospholipase-C catalyzed mechanism, occurring initially at the outer plasma membrane. In this report, I have further characterized this activity of IL-1. The hydrolysis of phosphatidylcholine was dependent upon extracellular Ca2+, although it appeared to be relatively independent of Mg2+. The activity was totally inhibited by prior treatment of intact Jurkat cells with trypsin. In addition, treatment of Jurkat cells with a phosphatidylinositol-specific phospholipase C, which selectively removes proteins anchored by glycosyl-phosphatidylinositol linkages, completely blocked the ability of IL-1 to stimulate the hydrolysis of phosphatidylcholine. These data suggest that the initial activity of IL-1 is to stimulate a Ca2+-dependent, glycosyl-PI-anchored phospholipase C, the active site of which is on the extracellular surface.
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PMID:Characterization of the interleukin-1-stimulated phospholipase C activity in human T lymphocytes. 281 73

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.
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PMID:Characterization of phosphoinositide-specific phospholipase C from human platelets. 282 91


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