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)

Crosslinking of the IgE receptor on the surface of rat basophilic leukemia (RBL) cells by multivalent antigen induces an association of these receptors with the detergent-insoluble membrane skeleton. Detergent insolubility of the receptor can also be induced on purified plasma membranes isolated from RBL cells by the use of either IgE oligomers or IgE monomers plus multivalent antigen. The critical event in initiating this interaction between the receptor and the membrane skeleton is cross-linking of the receptor. This association is rapid, and, when triggered by multivalent antigen, it is quickly reversed by the addition of excess monovalent antigen. The fact that this association occurs with the use of purified plasma membranes indicates that all of the components necessary for this interaction are present in the plasma membrane and that intracellular components are not required. Although crosslinking of the receptor activates phospholipase C and phospholipase A2 leading to the generation of several second messengers, none of these signaling mechanisms appears to be involved in IgE receptor interaction with the membrane skeleton. This interaction cannot be induced by phorbol 12-myristate 13-acetate (PMA), ionomycin, or a combination of these two reagents, although this will result in degranulation. Furthermore, receptor detergent insolubility is temperature independent when triggered by multivalent antigen, thus indicating that enzyme-catalyzed reactions are not important. This was verified by the fact that a variety of inhibitors that block phosphatidylinositol metabolism, arachidonic acid metabolism, Ca2+ influx, and protein kinase C (PKC) activation had no effect on antigen-induced association of the receptor with the membrane skeleton. These results indicate that the signaling mechanisms leading to the degranulation response are not involved in the association of the crosslinked receptor with the membrane skeleton.
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PMID:Association of the crosslinked IgE receptor with the membrane skeleton is independent of the known signaling mechanisms in rat basophilic leukemia cells. 183 Apr 93

In RBL-2H3 rat basophilic leukemia cells, Fc epsilon R1 crosslinking by multivalent antigen stimulates phosphatidylinositol (PI) turnover and Ca2+ influx and causes functional responses that include secretion, membrane ruffling and actin polymerization. Here, we show that the tyrosine kinase inhibitor, genistein, inhibits antigen-induced PI turnover, determined from assays of 1,4,5-inositol trisphosphate production, and impairs receptor-mediated secretion, ruffling and actin polymerization. Genistein has little effect on several functional responses to stimuli that bypass PI hydrolysis (ionomycin-induced secretion, phorbol ester-induced ruffling) but it inhibits phorbol ester-induced actin polymerization. These data implicate a common tyrosine kinase-dependent event, most likely the activation of phospholipase C gamma, in the Fc epsilon R1-mediated stimulation of PI turnover, secretion and ruffling. There may be additional tyrosine kinase-mediated events in the actin assembly pathway.
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PMID:Tyrosine kinase-dependent phosphatidylinostiol turnover and functional responses in the Fc epsilon R1 signalling pathway. 183 80

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.
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PMID:Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells. 184 42

Receptors for the chemotactic peptide fMet-Leu-Phe (fMet, N-formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL-60) cells and stimulate phospholipase C via a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein(s) [G-protein(s)]. We have developed methods for the assessment of formyl-peptide-receptor-stimulated binding of radiolabeled guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) to native HL-60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal (less than or equal to 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [35S]GTP[S] binding when GDP or GTP were present in high (greater than 100-fold) excess of [35S]GTP[S]. The increase in [35S]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparison of the number of chemotactic-peptide-sensitive [35S]GTP[S]-binding sites to the number of chemotactic peptide receptors present in HL-60 membranes provided direct evidence that a single formyl-peptide receptor is capable of catalyzing the binding of [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.
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PMID:Signal amplification in HL-60 granulocytes. Evidence that the chemotactic peptide receptor catalytically activates guanine-nucleotide-binding regulatory proteins in native plasma membranes. 190 7

Two murine monoclonal antibodies have been produced which identify a novel surface antigen expressed on human leucocytes in a non-lineage-restricted distribution. Antibodies WM-63 and WM-68 were derived after immunization of mice with human T-CLL cells and the leukaemic cell line HSB-2. Both antibodies were shown to react with over 90 per cent of normal T and B lymphocytes from peripheral blood and tonsil, and also with monocytes from peripheral blood. A subset of bone marrow leucocytes, including granulocyte-macrophage progenitors, were also reactive. No activity with non-haemopoietic cells or tissues could be identified, however WM-63 and WM-68 showed binding to virtually all cases of chronic B cell malignancy, including chronic lymphatic leukaemia and non-Hodgkin's lymphoma, as well as a proportion of cases of acute leukaemia. Although the antigen recognized by these antibodies could not be immunoprecipitated from membrane extracts, it was removed from the surface of intact cells using the proteolytic enzymes protease and papain. Re-expression on cultured cells was inhibited by incubation with puromycin, cycloheximide, and tunicamycin, indicating that the epitopes detected by WM-63 and WM-68 are likely to be carbohydrate moieties on a protein backbone. Removal of the antigen from the cell surface by treatment with the enzyme phosphatidyl-inositol phospholipase C indicates that it is linked by a phosphatidyl-inositol bond. WM-63 and WM-68 were both recently clustered at the Fourth International Workshop on Human Leucocyte Differentiation Antigens into CD-48, together with four other monoclonal antibodies. Although no biological function has been ascribed to the molecule detected by these antibodies, its restriction to the haemopoietic lineage suggests a role in regulation of leucocyte function.
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PMID:A novel non-lineage antigen on human leucocytes: characterization with two CD-48 monoclonal antibodies. 208 34

As reported by other workers, the treatment of rat basophilic leukemia RBL-2H3 cells with dexamethasone resulted in a marked decrease in responsiveness to Ag-stimulation. All responses measured, which included hydrolysis of inositol phospholipids, increase in concentration of cytosolic Ca2+, release of arachidonic acid and the secretion of serotonin, were suppressed, but once the cells were permeabilized the inhibitory actions of dexamethasone were no longer apparent. This suggested that all the necessary components of the stimulatory cascade were intact in the dexamethasone-treated cells. The measurement of phospholipase C activity in cell extracts and studies with phorbol ester also indicated that the cells contained a normal complement of phospholipase C and protein kinase C activity. We had previously shown that both Ag and the adenosine analog, 5'-(N-ethylcarboxamide)-adenosine, can activate phospholipase C, but they do so through different G proteins. Interestingly, the activation of phospholipase C by 5'-(N-ethylcarboxamide)-adenosine and the ensuing stimulatory events were markedly enhanced in dexamethasone-treated cells. The treatment with dexamethasone thus did not result in direct suppression of effector systems, but instead resulted in the selective modulation of the coupling between receptors and the effector systems by mechanisms that require soluble cytosolic factors.
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PMID:Treatment with dexamethasone down-regulates IgE-receptor-mediated signals and up-regulates adenosine-receptor-mediated signals in a rat mast cell (RBL-2H3) line. 213 82

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.
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PMID:On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins. 213 56

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-[beta-thio]diphosphate. ATP, adenosine 5'-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.
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PMID:Guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint. 217 6

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.
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PMID:Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells. 243 66

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b,k) and controlled by a gene locus closely linked to the Akp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i.e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78,000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing the Ly-31 and Akp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.
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PMID:Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone. 246 81


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