Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A naturally secreted protein, tissue inhibitor of metalloproteinases (TIMP), has been transiently expressed on the surface of transfected COS cells and stably on transfected murine BW 5147 thymoma cells, by linkage of the entire coding sequence of the cDNA to the last exon of Thy-1. Thy-1 is a glycophospholipid-linked protein. In COS cells the chimaeric protein can be labelled by [3H]ethanolamine, which is a component of glycophospholipid anchors. Ltk- cells cannot anchor proteins by glycan phosphatidylinositol linkage and were found to be unable to express the engineered protein extracellularly on their plasma membranes. Phosphatidylinositol-specific phospholipase C treatment released 90% of the protein from all BW 5147 cells, but very little from the COS-1 cells. It is concluded that the last exon of Thy-1 has conferred the property of glycophospholipid anchorage on the normally secreted protein TIMP.
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PMID:A cDNA construct of tissue inhibitor of metalloproteinases (TIMP) linked to the last exon of Thy-1 confers glycophospholipid anchorage on this naturally secreted protein. 134 44

The specific binding of radiolabeled polymyxin B (PmB) to rat alveolar macrophages was investigated. PmB retained its ability to inhibit lipopolysaccharide-induced tumor necrosis factor production by macrophages as long as one of five amino groups on PmB was unbound. Binding was saturable and temperature- and time-dependent, reaching steady state by 30 min at 37 degrees C and by 18 h at 4 degrees C. Macrophages had approximately 1.6 X 10(7) (Kd = 0.28 nM) PmB binding sites per cell. Lipid A had no appreciable effect on the number of sites. Binding did not occur to rat platelets, L929 fibroblast cells, a rat thymoma cell line, or precursor monocytic and myeloid cell lines. Precursor cells activated with 12-O-tetradecanoylphorbol-13-acetate acquired binding similar to that seen in alveolar macrophages, but L929 fibroblasts did not. Binding sites were sensitive to trypsin but not to phospholipase C. PmB may interact with specific binding sites involved in lipopolysaccharide-induced activation, production, or release of tumor necrosis factor by macrophages, inhibiting the effects of lipopolysaccharide on macrophages.
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PMID:Binding of polymyxin B to rat alveolar macrophages. 216 1

Cell surface antigens thought to be linked to the membrane via phosphatidylinositol (PI) are incompletely, and variably, released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The basis for this was investigated with cloned tumor cell lines and PI-PLCs isolated from two species of bacteria. Residual Thy-1 antigen, which was detectable by flow cytometry, remained on all thymoma cell lines after exposure to very high concentrations of either purified enzyme. A majority of the presumptive PI anchored molecules on all of the cell lines was sensitive to release by PI-PLC derived from Bacillus thuringiensis. However, cell lines differed dramatically in the ease with which PI-PLC from Staphylococcus aureus liberated the same surface antigens. This heterogeneity was determined at the single cell level because at least five different PI-anchored antigens exhibited similar behavior on a given cell line or transfected subclones of it. The two phospholipases differed with respect to molecular mass, serological cross-reactivity and sensitivity to inhibition by NaCl and detergents. These observations suggest that the two types of PI-PLC may have distinct substrate specificities or sensitivities to environmental conditions which account for the difference in their ability to act on PI-anchored proteins in particular cell types. Such enzymes should continue to be important tools for investigating the method and significance of attachment of lymphocyte surface glycoproteins. In particular, the S. aureus PI-PLC can be used to demonstrate and investigate a previously unrecognized heterogeneity in cells which express PI-anchored molecules.
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PMID:Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C. 245 50

The attachment of glycolipid anchors to the Thy-1 glycoprotein during biosynthesis was followed by the change of detergent-binding properties of biosynthetically labelled Thy-1 precursors upon phospholipase C treatment in the murine thymoma lines BW5147 and S1A. In S1A, 80% of the Thy-1 molecules were phospholipase-C-sensitive after a 2 min pulse with [35S]methionine, indicating that these molecules were already anchored via a glycolipid tail. In BW5147, 47% of the Thy-1 molecules had phospholipase-C-sensitive anchors attached after a 1.5 min labelling and, with longer pulses, this percentage rose to 76%. Tunicamycin did not block the addition of glycolipid anchors, and glycolipid attachment also occurred at 21 degrees C. The findings suggest that the attachment of glycolipid anchors occurs in the rough endoplasmic reticulum.
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PMID:Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. 289 53

We reported previously that the Thy-1 antigen was released from murine thymocytes and thymoma cells by S. aureus-derived phosphatidylinositol-specific phospholipase C (PI-PLC). It is therefore part of a small group of proteins known to use a unique form of membrane attachment. This finding has now been extended in studies with peripheral lymphocytes and additional leukocyte markers. Retention of viability and responsiveness to LPS were excellent in PI-PLC-treated spleen cells and there was no appreciable effect on lectin-binding surface glycoproteins. Thy-1 regeneration was insignificant on unstimulated spleen cells within 24 hr of treatment, but nearly complete at this time with a continuously dividing cell line. In contrast to the result with LPS, responses to the mitogens Con A, PHA, and PWM were virtually eliminated. Of more than 40 monoclonal antibodies tested, only staining with ThB and particular Qa specificities were diminished by PI-PLC treatment. The latter included Qa-2, Qa-4, Qa-5, and possibly also Qa-6, whereas Qa-1, TLa, and other class I and class II histocompatibility antigens were unaffected. Although the validity of the Qa results seems assured by the total PI-PLC resistance of many other lymphocyte antigens, the pattern of release was notably different from that observed with Thy-1 and ThB. That is, the density of Qa-2 was usually unchanged on a subpopulation of Qa-2-positive cells. This raises interesting questions about lymphocyte heterogeneity and flexibility in the use of this form of surface protein anchoring. Glycosyl-phosphatidylinositol-linked proteins may be functionally significant in immunological responses, and this experimental approach should continue to be valuable for their identification and characterization.
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PMID:Removal of lymphocyte surface molecules with phosphatidylinositol-specific phospholipase C: effects on mitogen responses and evidence that ThB and certain Qa antigens are membrane-anchored via phosphatidylinositol. 295 93

The rat T cell alloantigen RT6.2 shows a slow rate of synthesis in isolated T cells which hampers studies on the metabolism of RT6.2 in these cells (1). In order to facilitate further molecular and functional characterization of this molecule we have established a T-T hybridoma cell line which stably expresses RT6.2. Only 1 of 102 T cell hybridomas obtained upon fusion of the rat thymoma C58NT with DA rat lymph node cells expressed this antigen. This clone--EpD3--initially showed a relatively slow rate of cell division and a highly unstable pattern of expression of RT6.2. Thus, the relative number of RT6.2 bearing cells consistently decreased during cultivation of EpD3 and of EpD3-derived subclones. Cell separation studies suggest that the switch in RT6.2 phenotype of EpD3 cultures was due to the appearance of variant cells of RT6.2- phenotype with a higher proliferative capacity and was not induced by factors in the culture medium. By repeated panning and subcloning procedures to select for RT6.2 expressing cells, a subline of EpD3--EpD3/87--was obtained which stably expresses high levels of RT6.2. Metabolic labeling studies of RT6.2 in EpD3 show that it is synthesized very efficiently in these cells and support our previous suggestion that RT6.2 does not bear any classic oligosaccharide side chains. Moreover, RT6.2 can be released almost completely from EpD3 cells by phosphatidylinositol-specific phospholipase C (PI-PLC).
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PMID:Production of a rat T cell hybridoma that stably expresses the T cell differentiation marker RT6.2. 326 67

Proteins anchored in the membrane by covalent linkage to phosphatidylinositol (PtdIns) can be released by treatment with purified PtdIns-specific phospholipase C (Ptd-Ins-PLC). A recent survey of leukocyte antigens using flow cytometry has shown that staining of certain Qa antigens was diminished after PtdIns-PLC treatment, but staining of structurally related H-2 antigens was not affected. Therefore, in this study, the sensitivity of cell-surface Qa-2, H-2Kb, and H-2Db to hydrolysis by PtdIns-PLC was investigated biochemically by immunoprecipitation of radioiodinated molecules from cell lysates or supernatants. Qa-2, but not H-2Kb, was released from the surface of PtdIns-PLC-treated C57BL/10 mouse spleen cells and recovered in the cell supernatants. Similar analysis of thymoma cells transfected with cloned C57BL/10 genes showed that cell-surface Qa-2 molecules encoded by a Q7b cDNA and the Q7b or Q9b gene were sensitive to hydrolysis by PtdIns-PLC, whereas the H-2Kb and H-2Db gene products were resistant. Using thymoma cells transfected with hybrid genes constructed by exchanging exons between Q7b and H-2Db, the signals for PtdIns modification were localized to a defined region of Qa-2. This region differs from H-2Db most significantly by the presence of a central aspartate residue in the transmembrane segment and in the length of the cytoplasmic portion.
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PMID:Molecular mapping of signals in the Qa-2 antigen required for attachment of the phosphatidylinositol membrane anchor. 342 41

The biological significance of the action of glycosylphosphatidylinositol (GPI)-anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI-anchored proteins play a crucial role in the recently defined heavy metal (HgCl2)-triggered signal delivery to T lymphocytes. Thiol-reactive HgCl2, a multi-potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy-1, a representative GPI-anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl2 is diminished by the pre-treatment of cells with phosphatidylinositol-specific phospholipase C, which partially cleaved GPI-anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI-anchored proteins in the HgCl2-mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan-class A gene (PIG-A), and its restoration in a transfectant with PIG-A.
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PMID:Direct evidence of involvement of glycosylphosphatidylinositol-anchored proteins in the heavy metal-mediated signal delivery into T lymphocytes. 769 41

Glycosylated phosphoinositides serve as membrane anchors for numerous eukaryotic cell surface glycoproteins. Recent biochemical and genetic studies indicate that the glycolipids are assembled by sequential addition of components (monosaccharides and phosphoethanolamine) to phosphatidylinositol. The biosynthetic steps are presumed to occur in the ER, but formal proof of this is lacking. We describe experiments designed to establish the subcellular location of the initial steps in glycosyl-phosphatidylinositol (GPI) anchor biosynthesis and to define the transmembrane distribution of early biosynthetic lipid intermediates. The experiments were performed with the thymoma cell line BW5147.3. A subcellular fractionation protocol was used to show that early biosynthetic steps in GPI assembly, i.e., synthesis and deacetylation of N-acetylglucosaminyl phosphatidylinositol, occur in the ER. GPI biosynthetic intermediates were synthesized by incubating the microsomes with UDP-[3H]GlcNAc, and the transmembrane distribution of the labeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). Treatment of the radiolabeled microsomes with PI-PLC showed that > 70% of the N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be hydrolyzed, indicating that the two lipids were primarily distributed in the cytoplasmic (outer) leaflet of the microsomes. Similar cleavage results were obtained using Streptolysin O-permeabilized thymoma cells. When permeabilized cells were incubated with UDP-[3H]GlcNAc and treated with PI-PLC, approximately 85% of the radiolabeled N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be cleaved, indicating that they were accessible to the enzyme. The cumulative data indicate that early GPI intermediates are primarily located in the cytoplasmic leaflet of the ER, and are probably synthesized from PI located in the cytoplasmic leaflet and UDP-GlcNAc synthesized in the cytosol.
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PMID:Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer. 850 Nov 24

Paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) is a putative autocrine survival factor for the preimplantation embryo. It acts to induce receptor-mediated calcium transients in the early embryo. Inhibitors of 1-o-phosphatidylinositol-3-kinase (PI3kinase), such as wortmannin and LY 294002, blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in autocrine signal transduction in the early embryo. Perfusion of the embryo cytoplasm with a blocking antibody to PIP3 inhibited paf-induced calcium transients and hyperpolarization of the membrane potential. Furthermore, direct infusion of PIP3 into the embryo induced a nifedipine (10 micromol/L)- and diltiazem (10 micromol/L)-sensitive calcium current in the 2-cell embryo. PIP3 acts as a docking site on membranes for proteins that contain pleckstrin homology domains, such as the thymoma viral proto-oncogene protein (AKT) and phospholipase C gamma. The 2-cell embryo expressed three genes for AKT (Akt 1-3) and two genes for phospholipase C gamma (Plcg1 and Plcg2), and we confirmed the expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced increased accumulation of serine 473-phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane PIP3. Inhibitors of PI3kinase, such as LY294002, and of AKT, e.g., deguelin and AKT-inhibitor, reduced zygote development in a dose-dependent manner, and this inhibition was partially reversed by the addition of paf to the culture medium. These results provide the first direct evidence that PIP3 and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via PI3kinase has important roles in governing the cell survival pathways, these results support the hypothesis that autocrine embryotropins, such as paf, act as survival factors.
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PMID:Direct evidence for the action of phosphatidylinositol (3,4,5)-trisphosphate-mediated signal transduction in the 2-cell mouse embryo. 1763 44


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