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)

There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration. These results strongly support the hypothesis that in HL-60 granulocytes, PEt is formed via PLD-catalyzed transphosphatidylation. Moreover, using HL-60 granulocytes double-labeled by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC, it has been established that the early (30 s) appearance of alkyl-PA is due primarily to PLD, not phospholipase C as previously thought, and that alkyl-PEt is formed exclusively by PLD. These results constitute the first direct evidence for receptor-linked activation of PLD, leading to the generation of PA and PEt in an intact cell system.
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PMID:Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. 316 77

Using the [3H]inositol-labeled plasma membranes isolated from the differentiated human leukemic (HL-60) cells, the mode of inhibitory action of the Ca2+/phospholipid-dependent enzyme protein kinase C in the chemotactic peptide, fMet-Leu-Phe (fMLP)-induced, phospholipase C-mediated hydrolysis of phosphoinositides was investigated. In this cell-free membrane system, fMLP in the presence of GTP plus Ca2+, GTP in the presence of Ca2+, or Ca2+ alone could induce the formation of inositol bis- and trisphosphate (IP2 and IP3, respectively). When the intact cells were pre-treated with 12-O-tetradecanoylphorbol-13-acetate, the fMLP- and GTP-induced formation of IP2 and IP3 was markedly reduced but the Ca2+-induced reactions were not reduced in the isolated membranes. This result suggests that protein kinase C impairs the coupling of the GTP-binding protein to the phospholipase C. In another experiment, preincubation of the isolated membranes with pure rat brain protein kinase C inhibited the fMLP-induced formation of IP2, but did not inhibit the GTP- or Ca2+-induced reaction. Under the same conditions, protein kinase C did not inhibit the fMLP-, GTP-, or Ca2+-induced formation of IP3. This result suggests that protein kinase C impairs additionally the coupling of the fMLP receptor to the GTP-binding protein leading to the formation of IP2. The reason for the failure of protein kinase C to inhibit the fMLP-induced formation of IP3 in the cell-free membrane system is unknown, but several possible mechanisms are discussed.
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PMID:Modes of inhibitory action of protein kinase C in the chemotactic peptide-induced formation of inositol phosphates in differentiated human leukemic (HL-60) cells. 347 61

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.
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PMID:Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils. 349 Oct 71

Rabbit neutrophils labelled with [3H]inositol and permeabilized with saponin produced [3H]inositol trisphosphate (InsP3) when incubated with stable analogues of GTP or millimolar concentrations of Ca2+. [3H]InsP3 production elicited by guanosine 5'-[gamma-thio]triphosphate was enhanced by the chemoattractant formylmethionyl-leucyl-phenylalanine and inhibited by pertussis-toxin pretreatment. A pertussis-toxin-sensitive stimulation of [3H]InsP3 concentration was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with guanosine 5'-[beta-thio]diphosphate or GTP. Millimolar Ca2+ alone was sufficient to stimulate [3H]InsP3 production; however, in the presence of guanosine 5'-[gamma-thio]triphosphate, the Ca2+ dose-response curve was shifted to submicromolar concentrations. These findings directly confirm the role of a pertussis-toxin-sensitive guanine nucleotide regulatory protein (G protein) in chemoattractant-stimulated phospholipase C activity in rabbit neutrophils. Moreover, the ability of guanine nucleotides to sensitize phospholipase C to physiologically relevant Ca2+ concentrations suggests that the role of the activated G protein may be to enhance the apparent affinity of phospholipase C for Ca2+ and thus to activate the enzyme without an increase in the Ca2+ concentration.
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PMID:Guanine nucleotide regulation of phospholipase C activity in permeabilized rabbit neutrophils. Inhibition by pertussis toxin and sensitization to submicromolar calcium concentrations. 354 23

The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.
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PMID:Chemotactic peptide activation of human neutrophils and HL-60 cells. Pertussis toxin reveals correlation between inositol trisphosphate generation, calcium ion transients, and cellular activation. 387 77

Various n-formylated peptides function as receptor-specific chemoattractants for both granulocytes and monocytes. Because these agents are important tools in the study of leukocyte function in vitro, we chose to examine their effects on leukocyte procoagulant activity. The synthetic chemotactic peptide N-formyl-methionyl-leucyl phenylalanine (FMLP) induces a fourfold increase in procoagulant activity (PCA) in cultured human monocytes at an optimal dose of 5 X 10(-9) mol/L, whereas higher doses inhibit PCA response. Although nonadherent lymphocytes are not absolutely required for PCA expression, their presence significantly amplifies monocyte PCA. Irradiation of nonadherent lymphocytes before mixing them with FMLP and adherent cells abolishes their ability to amplify PCA. Kinetic studies demonstrate an increase in optimal dose FMLP-stimulated PCA over time whereas high-dose inhibition of PCA generation occurs at various incubation times. Cell viability is unaffected by inhibitory concentrations of FMLP. Supernates from high-dose FMLP-stimulated cells fail to inhibit later expression of PCA by cells exposed to endotoxin. The cellular procoagulant remains cell-bound and exhibits characteristics of thromboplastin (tissue factor), including inhibition by concanavalin A and phospholipase C as well as the ability to shorten the clotting times of factor VIII but not factor VII-deficient substrate plasmas. These results suggest a complex system of lymphoid cell regulation of procoagulant generation by monocytes exposed to various chemotactic peptides in vitro.
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PMID:Regulation of monocyte procoagulant by chemoattractants. 397 Oct 40

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.
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PMID:Secretagogue-induced phosphoinositide metabolism in human leucocytes. 608 66

Incubation of human neutrophils with phospholipase C from Clostridium perfringens caused an increase in the ability of the treated cells to bind the chemotactic peptide, F-Met-Leu-Phe. The increase in binding was related to an increase in specific binding of the ligand. The increase in specific binding was, in turn, related to an increased number of peptide receptors. The dissociation constant (KD) for the tripeptide was not altered, on the average, by enzyme treatment. The increase in peptide receptor number was related temporally, and possibly mechanistically, to enzyme-stimulated secretory function involving the secondary granules. Phospholipase C treatment did not similarly augment binding of the complement-derived attractant, C5a. Receptor numbers for different chemotactic ligands may therefore be controlled by different mechanisms. Supplementary experiments provided evidence that this phenomenon was attributable to phospholipase C activity and not to contaminating protease(s).
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PMID:Human neutrophil peptide receptors: mobilization mediated by phospholipase C. 628 27

Human and rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and A23187 show a loss of phosphatidylinositol and an increase in phosphatidate. In cells prelabelled with 32Pi it would be expected that the newly synthesised phosphatidate would have the same specific activity as cellular ATP, provided that the loss of phosphatidylinositol is by phospholipase C attack and the resultant diacyglycerol is phosphorylated by ATP. Instead, it is demonstrated that the specific activity of newly-formed phosphatidate is less than a tenth of the specific activity of ATP initially followed by a gradual increase. The time-course of mass and of [3H]glycerol-labelled phosphatidate formation (from cells pulse-labelled with [3H]glycerol) is similar to enzyme release but differs from the generation of 32P-labelled phosphatidate (from cells prelabelled with 32Pi). The source of the newly formed phosphatidate is most likely from phosphatidylinositol because: (a) The increase in phosphatidate is always accompanied by a loss of phosphatidylinositol with no changes in the other lipids. (b) Cells pulse-labelled with [3H]glycerol lose label from phosphatidylinositol only and this is accompanied by an increase in label in phosphatidate. (c) The specific activity of the newly synthesised phosphatidate is closest to the specific activity of phosphatidylinositol. One plausible explanation for these results is that phosphatidylinositol is directly converted to phosphatidate by phospholipase D action and the resulting phosphatidate accumulates radioactivity by exchange of its phosphate group with ATP. It is also shown that enzyme secretion and conversion of phosphatidylinositol to phosphatidate can depend on both intra- as well as extracellular Ca2+. Depletion of the intracellular pool of Ca2+ is essential to inhibit totally the enzyme secretion and the conversion of phosphatidylinositol to phosphatidate in agreement with our previous results on rabbit neutrophils (Cockcroft, S., et al. (1981) Biochem. J. 200, 501-508).
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PMID:Ca2+-dependent conversion of phosphatidylinositol to phosphatidate in neutrophils stimulated with fMet-Leu-Phe or ionophore A23187. 643 54

The effect of staphylococcal alpha-toxin on the chemotactic response of human polymorphonuclear leukocytes was studied by measuring the migration of alpha-toxin-treated cells either through membrane filters toward C5a or under agarose toward N-formyl-l-methionyl-l-phenylalanine. At doses of greater than or equal to 5 hemolytic units, alpha-toxin depressed chemotactic responsiveness in both best systems. Further studies revealed that alpha-toxin was also a potent granulocyte aggregant at doses similar to those necessary for depressed chemotactic capacity. It is proposed that the inhibitory effect of this membrane-active toxin on chemotactic function may be related to increased granulocyte adhesiveness and that the pathogenic properties of alpha-toxin may in part by explained by these effects.
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PMID:Effect of staphylococcal alpha-toxin on neutrophil migration and adhesiveness. 732 97


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