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)

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.
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PMID:Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes. 131 77

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the major phosphoinositide-specific phospholipase C of human amnion. 196 96

The coupling of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li+ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium +/- inositol (40 microM). In inositol-replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0-30 min) approximately > or = 20-fold activation of phospholipase C, whereas prolonged (> 60 min) stimulation turned over PtdIns equal to the cellular total mass, involving approximately 80% of the cellular PtdIns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC50 approximately 20 microM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol-replete or depleted cells basal concentrations of inositol and CMP-phosphatidate were respectively approximately 20 mM or < or = 100-500 microM and approximately 0.1 or approximately > or = 1-10 pmol/mg of protein. Comparison of the effects of agonist +/- Li+ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP-phosphatidate or inositol in inositol-replete or depleted cells, respectively. Thus, the preferential capacity of Li+ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.
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PMID:The mechanism of muscarinic receptor-stimulated phosphatidylinositol resynthesis in 1321N1 astrocytoma cells and its inhibition by Li+. 759 17

Membranes from human brain cortex (8-12 h post mortem) were labelled with [3H]inositol, in the presence of CMP, through the back reaction catalysed by PtdIns synthase. The enzyme incorporated [3H]inositol into phosphoinositides at a maximal rate of 419 pmol min-1 mg protein-1. In the absence of CMP, the labelling rate due to the PtdIns headgroup exchanging enzyme was 36 pmol min-1 mg protein-1. Human brain PtdIns synthase showed Kmapp values of 0.49 mM and 18 microM for inositol and CMP, respectively. In the presence of ATP, [3H]polyphosphoinositides formed after [3H]PtdIns were hydrolysed by phospholipase C in a GTP gamma S and neurotransmitter receptor agonist-dependent manner. Production of 3H-inositol phosphates as stimulated by GTP gamma S (350% of basal) was increased by the muscarinic agonists carbachol and oxotremorine-M (600% of basal) and by serotonin (485% of basal). The relative potencies of carbachol and oxotremorine-M were consistent with an action at muscarinic receptors. These results show that coupling between muscarinic and serotonin receptors and phospholipase C is preserved in membranes from post mortem human brain cortex and validate the use of a method involving direct [3H]inositol labelling of a membrane fraction to study the functional state of phospholipase C-coupled receptors in human brain samples.
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PMID:Neurotransmitter-stimulated breakdown of endogenous polyphosphoinositides in post mortem human brain. 791 14

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.
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PMID:The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells. 811 Jan 90

We investigated the relationship between substrate aggregation and activation of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1), isolated from bovine brain cytosol. The inositol lipids 1,2-dibutyryl-sn-glycero-3-phosphoinositol (di-C4-PI), 1,2-dihexanoyl-sn-glycero-3-phosphoinositol (di-C6-PI), and 1,2-dioctanoyl-sn-glycero-3-phosphoinositol (di-C8-PI) were prepared from synthetic cytidine diphosphate diglyceride analogs in a reaction with myo-inositol catalyzed by yeast phosphatidylinositol synthase. All three lipids served as substrates for PLC-delta 1 at concentrations significantly below their critical micelle concentration (cmc). Under these conditions, steps that might limit the reaction rate, such as membrane adsorption or penetration into the phospholipid surface, were eliminated. Below the cmc, the concentration of lipid substrate required to produce hydrolysis followed the order: di-C8-PI < di-C6-PI << di-C4-PI. Calcium was essential for hydrolysis of the short chain substrates at all lipid concentrations tested. The dependence of the reaction on calcium suggests that this ion activates PLC-delta 1 at a step other than adsorption to or penetration of the membrane surface. As the concentration of di-C8-PI was raised above the cmc, the reaction velocity increased 2-3-fold. These results are consistent with the idea that micellar or bilayer aggregates of phosphoinositol are not required for PLC-catalyzed hydrolysis, although the reaction rate is enhanced by micelle formation.
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PMID:Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1. 838 May 75

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.
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PMID:Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes. 839 10

Phosphatidylinositol (PI) turnover is considered to be involved in the regulation of cell growth. The enzymes for PI turnover include phospholipase C (PLC), PI4-kinase and PI synthase. We have isolated pholipeptin and fluvirucin B2 from microorganisms and akaterpin from a marine sponge as PLC gamma inhibitors. We also isolated echiguanines from Streptomyces as PI4-kinase inhibitors. Since echiguanines did not inhibit the enzyme in situ, we synthesized their ribosylated derivatives that were effective in cultured cells. We previously isolated inostamycin from Streptomyces as an inhibitor of PI synthase. We found that inostamycin induced G1 block in cycling NRK cells. Inostamycin inhibited the serum-induced S-phase induction in quiescent NRK cells. Inostamycin was found to decrease serum-induced expression of cyclin D and cyclin E, without inhibiting the activation of MAP kinase. It also inhibited serum-induced activation of CDK2 and phosphorylation of pRB. Thus, PI synthesis was suggested to be involved in regulation of serum-induced S-phase induction by modulating G1 cyclin expression.
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PMID:[Screening of phosphatidylinositol turnover inhibitors and regulation of cell cycle progression]. 930 57

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy.
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PMID:Phosphatidylinositol synthesis in mycobacteria. 998 74

The effect of the adenosine (AD) analog 2-chloroadenosine (C-AD) on glucose-induced inhibition of phosphoinositide synthesis was studied in human retinal pigment epithelial (RPE) cells by monitoring the level of the phosphatidylinositol (PI) synthase substrate, cytidine diphosphate diglyceride (CDP-DG). In high-aldose reductase (AR)-expressing RPE 91 cells, C-AD decreased CDP-DG at 5 mmol/L glucose and reversed the increase by 20 mmol/L glucose. AD deaminase (ADA), which inactivates endogenously released AD, potentiated the hyperglycemia-induced increase in CDP-DG. Theophylline, an AD-A1 and AD-A2 receptor antagonist, caused an increase in CDP-DG at 20 mmol/L glucose. C-AD did not alter CDP-DG in low-AR-expressing RPE 45 cells, but did decrease CDP-DG after cells were conditioned in 300 mmol/L glucose for 1 week (which induces AR). The mechanism by which AD regulates PI synthase in cells with high AR activity is unknown, but it is independent of Gi or Gs proteins, adenylate cyclase and phospholipase C (PLC) activation, myo-inositol (MI) uptake, or MI efflux. Administration of C-AD to streptozotocin-induced diabetic rats prevented the slowing of motor nerve conduction velocity (MNCV). Thus, AD derivatives, which reverse a glucose-induced deficit in phosphoinositide metabolism, might serve as a useful pharmacological tool to intervene in hyperglycemia-induced diabetic complications.
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PMID:2-Chloroadenosine reverses hyperglycemia-induced inhibition of phosphoinositide synthesis in cultured human retinal pigment epithelial cells and prevents reduced nerve conduction velocity in diabetic rats. 1042 Dec 20


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