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)

We showed previously that glomerular mesangial cells displayed increased fibronectin, laminin, and type IV collagen synthesis and mRNA levels when grown in medium containing 30 mM glucose compared with those cells grown in 10 mM glucose [S. H. Ayo, R. A. Radnik, W. F. Glass II, J. A. Garoni, E. R. Rampt, D. R. Appling, and J. I. Kreisberg. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F185-F191, 1990]. However, total protein synthesis and actin mRNA were unchanged. In this report, we show that an increase in medium glucose concentration resulted in an increase in diacylglycerol (DAG) mass and transiently increased protein kinase C (PKC) activity as assessed by the translocation of PKC from the soluble to the particulate fraction. Effects of increased glucose on DAG were evident at 30 min and were maintained through 1 wk of growth in medium containing 30 mM glucose. Although total PKC activity (i.e., soluble plus particulate fractions) did not change with high-glucose treatment, the percent activity associated with the particulate fraction (i.e., activated PKC) increased significantly after 60 min in RPMI 1640 medium with 30 mM glucose. The distribution of PKC returned to control values by 24 h. High glucose did not stimulate phosphoinositide hydrolysis, as evidenced by the absence of an increase in the water-soluble inositol phosphates, indicating that DAG was not generated through the action of a phosphoinositide-specific phospholipase C. Cells treated with the cell-permeable DAG analogue 1-oleoyl-2-acetyl glycerol to activate PKC displayed approximately two-fold increases of fibronectin, laminin, and type IV collagen mRNA levels after normalization against actin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures. 192 72

Murine T cells synthesize and express a cell-surface glycophospholipid anchored 40 kDa and a secreted water-soluble 39 kDa Qa-2 polypeptide. We have examined the biosynthetic pathways which lead to the production of the membrane-bound and water-soluble isoforms of the Qa-2 molecule. Using the detergent TX-114, both detergent (membrane)-bound and soluble Qa-2 polypeptides can be identified in cell lysates and can be distinguished by charge and molecular weight. Two membrane-bound forms, a 40-kDa Endo H resistant cell-surface form and a 38 kDa-Endo H sensitive form can be identified, both of which can be biosynthetically labeled with 3H-ethanolamine and can be converted to water soluble forms by digestion with a phosphatidylinositol specific phospholipase C. In addition, several water soluble polypeptides at 39, 37, 35 kDa, and a minor species at 33 kDa were identified, none of which radiolabel with 3H-ethanolamine. While the 39-kDa polypeptide was Endo H resistant, the other isoforms were sensitive to Endo H digestion. Pulse chase experiments and molecular weights of the deglycosylated core polypeptides suggest a precursor to product relationship between the intracellular water-soluble species and the mature 39-kDa secreted Qa-2 molecule. This relationship is supported by the observation that murine L cells transfected with the Qa-2 encoding class I gene Q7 fail to express membrane-bound Qa-2 molecules yet synthesize both intracellular water-soluble and secreted Qa-2 molecules. These findings argue for a pathway in which secreted soluble Qa-2 molecules are derived from intracellular precursors.
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PMID:Biosynthesis of glycophospholipid bound and secreted murine class I Qa-2 polypeptides. 196 Dec 2

Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.
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PMID:Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry. 199 39

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.
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PMID:Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts. 199 79

The activity of a phospholipase C or phospholipase D may be assessed by measuring the radioactivity or phosphate released into the aqueous phase of a lipid extract. However, in crude enzyme fractions, this type of analysis may not be possible due to formation of water-soluble metabolites by other enzymatic reactions, as demonstrated here with a crude lysosomal enzyme fraction. In such instances, analysis of both water-soluble and lipid-soluble metabolites, at various times of incubation, may still provide clear identification of phospholipases C or D, even when a variety of lipases and other hydrolases are present.
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PMID:Assay of phospholipases C and D in presence of other lipid hydrolases. 205 9

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.
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PMID:Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity. 211 61

Growth Hormone has recently been shown to stimulate the formation of diacylglycerol in Ob1771 mouse preadipocyte cells without increasing inositol lipid turnover. Addition of growth hormone to Ob1771 cells prelabelled with [3H]glycerol or [3H]choline led to a rapid, transient and stoechiometric formation of labelled diacylglycerol and phosphocholine, respectively. In contrast, no change was observed in the level of choline and phosphatidic acid whereas the release of water-soluble metabolites in [3H]ethanolamine prelabelled cells exposed to growth hormone was hardly detectable. Stimulation by growth hormone of cells prelabelled with (2-palmitoyl 9, 10 [3H])phosphatidylcholine also induced the production of labelled diacyglycerol. Pertussis toxin abolished both diacylglycerol and phosphocholine formation induced by growth hormone. It is concluded that growth hormone mediates diacylglycerol production in Ob1771 cells by means of phosphatidylcholine breakdown involving a phospholipase C which is likely coupled to the growth hormone receptor via a pertussis toxin-sensitive G-protein.
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PMID:Diacylglycerol production induced by growth hormone in Ob1771 preadipocytes arises from phosphatidylcholine breakdown. 212 19

The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol-specific phospholipase C (PtdIns-specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non-N-acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry. The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns-specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 micrograms carbohydrate caused 60% inhibition of the binding of anti-CRD antibodies raised against the soluble form of variant surface glycoprotein.
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PMID:Structural features of the lipopeptidophosphoglycan from Trypanosoma cruzi common with the glycophosphatidylinositol anchors. 214 55

In a recent report, a construction containing the alpha chain-variable region (V alpha) coding sequence of a cDNA clone derived from a diphtheria toxoid-specific human T cell (P28), fused to a human immunoglobulin kappa light chain constant region (Ck), was used stably to transfect a murine myeloma cell. In the present study, these transfected cells were employed as an immunogen to raise a mAb, termed 1C5V alpha, specific both for the V alpha Ck chimeric protein secreted by the transfectant and the P28 T cell antigen receptor-V alpha region. mAb 1C5V alpha specifically immunoprecipitates the V alpha Ck protein as a family of 32-35 kDa bands present in the 35S-methionine-labeled culture supernatant from the transfected cells. It specifically binds clone P28. Surface molecules recognized by mAb 1C5V alpha are physically linked to the CD3 molecules since cell treatment with either 1C5V alpha or anti-CD3 mAbs caused the simultaneous down-regulation of the CD3/TCR molecular complex. This link is further supported by immunoprecipitation experiments. Thus, both the 1C5V alpha and the anti-CD3 mAbs precipitate the 16-28 kDa CD3 molecules and the disulfide-linked form of P28 TCR from 125I-labeled P28 T cells. Studies performed in order to define whether a stimulus directly acting on the TCR-V alpha region may trigger the intracellular events observed during human T cell activation showed that (a) mAb 1C5V alpha efficiently triggers the phospholipase C transduction pathway revealed by an accelerated phosphoinositides turn-over and an increased production of phosphorylated derivatives of inositol phosphates; (b) mAb 1C5V alpha induces an up-regulation of IL2R mRNA, accompanied by a slight increase of IL2 and IFN alpha mRNA transcripts evidently amplified in the presence of PMA; (c) soluble mAb 1C5V alpha is strongly mitogenic together with PMA. These results provide the first evidence for the structural authenticity of a secreted water-soluble chimeric form of the variable region of a human TCR alpha chain. They further demonstrate that such chimeric proteins may be valuable tool to further dissect the various functional structure of the human TCR.
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PMID:A human TCR-Ig chimeric protein used to generate a TCR alpha chain variable region-specific mAb. 214 29

The metabolism and intracellular distribution of a fluorescent analog of phosphatidylinositol (PI), 1,2-[oleoyl,N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl) aminocaproyl)]-PI (C6-NBD-PI), was examined in monolayer cultures of Swiss 3T3 cells following its insertion into the plasma membrane. Evidence is presented that the exogenously supplied C6-NBD-PI was hydrolyzed by a calcium-dependent PI-specific phospholipase C (PI-PLC) at the external cell surface and that this PI-specific phospholipase C may play a role in the density-dependent inhibition of cell growth: (i) When confluent monolayer cultures were incubated with C6-NBD-PI for 60 min at 7 degrees C, the lipid spontaneously transferred to the cells, and prominent labeling of intracellular membranes was observed. Lipid extraction and analysis demonstrated that more than 60% of the fluorescent lipid in these cells was fluorescent diacylglycerol (DAG). However, when the corresponding fluorescent analogs of phosphatidylcholine or phosphatidylethanolamine were used, the fluorescent lipids readily transferred to cells, but no hydrolysis to fluorescent DAG occurred. (ii) Both intracellular labeling and hydrolysis of C6-NBD-PI to -DAG were inhibited in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. (iii) When myo-[2-3H]inositol-labeled C6-NBD-PI was incubated with cells, [3H]inositol phosphate was released into the incubation medium, but no water-soluble 3H-labeled products were found associated with the cells. (iv) The level of C6-NBD-PI hydrolysis increased dramatically with increasing density of 3T3 cells in monolayer culture.
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PMID:Detection of a phosphatidylinositol-specific phospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth. 215 13


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