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)

Arachidonic acid has been implicated as a second messenger in insulin secretion on the basis of (1) mobilization of intracellular Ca2+ from the endoplasmic reticulum of islets and (2) amplification of voltage-dependent Ca2+ entry. The insulin secretagogues D-glucose and the muscarinic agonist carbachol both increase unesterified arachidonic acid accumulation in isolated islets. We now show that diacylglycerol, a product of phospholipase C action, is a major source of free arachidonic acid in islets. Diacylglycerol hydrolysis in islets occurs through a two-step process. In the first step, the sn-1 bond of 1-stearoyl-2-arachidonyl-sn-glycerol is hydrolyzed by a diacylglycerol lipase, giving rise to 2-arachidonyl-sn-glycerol. Next, the sn-2 bond of 2-arachidonyl-sn-glycerol is hydrolyzed by a monoacylglycerol lipase, which is the rate-limiting step, releasing unesterified arachidonic acid. Both diacylglycerol lipase and monoacylglycerol lipase are highly enriched in the plasma membrane of beta-cells. Diacylglycerol lipase activity in islet homogenates is selectively inhibited in a dose-dependent manner by the compound RHC-80267, a specific diacylglycerol lipase inhibitor. RHC-80267 inhibits glucose- and carbachol-induced insulin release from intact islets in a dose-dependent manner that parallels its inhibition of diacylglycerol lipase activity. Importantly, RHC-80267, at concentrations that almost completely inhibit diacylglycerol lipase activity and glucose- and carbachol-induced insulin secretion by islets, markedly inhibits glucose- and carbachol-induced increases in islet arachidonic acid levels, as measured by gas chromatography with electron-capture detection of its pentafluorobenzyl esters. RHC-80267 did not significantly affect islet glucose oxidation, phospholipase C, monoacylglycerol lipase, or phospholipase A2. Since glucose and carbachol are known to stimulate phospholipase C, our observations indicate that diacylglycerol is an important source of arachidonic acid and other free fatty acids in islets. Furthermore, production of arachidonic acid from the hydrolysis of diacylglycerol is essential for glucose- and carbachol-induced insulin secretion.
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PMID:Diacylglycerol hydrolysis to arachidonic acid is necessary for insulin secretion from isolated pancreatic islets: sequential actions of diacylglycerol and monoacylglycerol lipases. 794 36

Stable clones of NIH 3T3 fibroblasts transfected with the cDNA of either the wild-type or deletion forms of the rat type I (or cerebellar) inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) were investigated. The delta form, missing the NH2-terminal sequence that includes the IP3-binding site, is expected to be still assembled with wild-type subunits to yield a tetrameric Ca2+ channel across the endoplasmic reticulum membrane; the s form, missing the membrane-spanning sequences, is expected to remain as a soluble monomer in the cytosol. With respect to control clones transfected with the vector only, the synthesis fo IP3Rs was markedly stimulated in the receptor-transfected clones. The mass accumulation, however, was increased only moderately (deletion forms = 15-30% of the endogenous IP3R), apparently because of a compensatory increase in receptor turnover. Coordinate changes in IP3 generation and Ca2+ release were revealed in the delta clones by experiments in both intact and permeabilized cells. In these clones, the IP3R was more sensitive to IP3, and IP3 generation at the ATP P2u surface receptor was decreased. This latter effect was due neither to a defect in G protein coupling nor to changes in phospholipase C expression, but to down-regulation of the P2u receptor. In the cells expressing the s- and delta-IP3R subunits, no differences with respect to the controls were observed in epidermal growth factor-induced DNA synthesis, whereas long-term growth stimulated by serum was reduced. Even more marked, especially in the delta clones (-90%), was the inhibition of cell transformation induced by autocrine stimulation with transforming growth factor alpha of the overexpressed epidermal growth factor receptors or by other growth factor receptors and oncogenes (platelet-derived growth factor/platelet-derived growth factor receptor beta, HER2/neu, and v-erbB). These effects appear not to be connected to the signaling processes mediated by tyrosine phosphorylation since the latter was unchanged in the delta clones. These results demonstrate for the first time (a) that the changes in cellular homeostasis directly induced by deleted IP3R subunits (increased receptor synthesis and increased IP3R sensitivity) are largely compensated by indirect coordinate changes apparently aimed to keep near normal the signaling properties of the cells; (b) that modulation of intracellular Ca2+ channels induces profound consequences that differentially affect growth and oncogenesis; and (c) that IP3Rs and the Ca2+ stores are important cross-roads of intracellular signaling pathways.
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PMID:Stable expression of truncated inositol 1,4,5-trisphosphate receptor subunits in 3T3 fibroblasts. Coordinate signaling changes and differential suppression of cell growth and transformation. 803 82

Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein.
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PMID:Topology of GPI biosynthesis in the endoplasmic reticulum. 808 Dec 26

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.
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PMID:GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways. 808 Dec 27

Using antibody raised against putative Form I phosphatidylinositide-specific phospholipase C (PI-PLC) and direct amino acid sequencing of the protein recognized by this antibody, we have shown that the antibody reacts with luminal endoplasmic reticulum (ER) proteins, including ERp61. ERp61 possesses a COOH-terminal QEDL sequence that acts as an ER retention signal. Additional experiments have shown, however, that PI-PLC activity is separable from ERp61 and that rat or murine ERp61 expressed in COS cells failed to produce an increase in PI-PLC activity in the COS cells. Finally, we have identified ERp61 as GRP58, a 58-kDa protein inducible by glycosylation block and treatment with the Ca2+ ionophore, A23187.
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PMID:Erp61 is GRP58, a stress-inducible luminal endoplasmic reticulum protein, but is devoid of phosphatidylinositide-specific phospholipase C activity. 810 75

Bone is a target tissue of androgens, but the mechanisms by which they act on bone are still unclear. This study examines the early (5-60 s) effects of 1 pM to 1 microM testosterone on cytosolic free Ca2+ concentration ([Ca2+]i) and inositol 1,4,5-trisphosphate (InsP3) and diacyglycerol (DAG) formation in confluent male rat osteoblasts. 10 pM to 10 nM testosterone increased [Ca2+]i within 5 s via Ca2+ influx as shown by the effects of EGTA and the Ca2+ channel blockers nifedipine and verapamil and via Ca2+ mobilization from the endoplasmic reticulum as shown by the effects of thapsigargin and neomycin. 10 pM to 10 nM testosterone increased InsP3 and DAG formation within 10 s. Testosterone immobilized on bovine serum albumin (testosterone (O-carboxymethyl)oxime/bovine serum albumin) and its derivative, (O-carboxymethyl)oxime, rapidly increased [Ca2+]i and InsP3 and DAG formation and were full agonists, although they were less potent than the free steroid. Cyproterone acetate, a nuclear antagonist, did not block the increase in [Ca2+]i and InsP3 and DAG formation induced by testosterone. Finally, neomycin and pertussis toxin totally abolished the effects of testosterone on InsP3 and DAG. These results suggest that male rat osteoblasts bear nongenomic unconventional cell-surface receptors for testosterone that belong to the class of the membrane receptors coupled to a phospholipase C via a pertussis toxin-sensitive G-protein.
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PMID:Androgens increase intracellular calcium concentration and inositol 1,4,5-trisphosphate and diacylglycerol formation via a pertussis toxin-sensitive G-protein. 812 34

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
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PMID:A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways. 813 15

We recently showed that when rats were administered the inhalation anesthetic halothane, a 58 kDa liver endoplasmic reticulum protein became covalently trifluoroacetylated by the trifluoroacetyl chloride metabolite of halothane. Although the 58 kDa protein showed 99% identity to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha, it did not have phosphatidylinositol-specific phospholipase C activity. It was concluded that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha actually encoded for the 58 kDa endoplasmic reticulum protein of unknown function. Other researchers have come to the same conclusion and have shown that the 58 kDa protein has protein disulfide-isomerase and protease activities. We now report that patients with halothane hepatitis have serum antibodies that react with both purified trifluoroacetylated and native rat liver 58 kDa proteins. These results suggest that when patients are exposed to halothane a human liver orthologue of the rat liver trifluoroacetylated-58 kDa protein is formed. In certain patients, this protein may become immunogenic and lead to the formation of specific antibodies and or specific T-cells, which may react with both trifluoroacetylated and native 58 kDa proteins, and ultimately be responsible, at least in part, for the hepatitis caused by halothane.
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PMID:Association of anti-58 kDa endoplasmic reticulum antibodies with halothane hepatitis. 821 76

A microsomal protein having N-terminal amino acid sequence SDVLELTDEN, was initially described as a phosphatidyl inositol-specific phospholipase C alpha when its cDNA was cloned (Bennett et al., Nature, 334, 268, 1988). Later, this protein, with an estimated molecular mass of 54 to 60 kDa, was shown to lack the phospholipase activity and instead a protein disulfide oxidoreductase and a thiol protease activities were ascribed to it. Following evidences indicated that the protein in question is the carnitine medium/long chain acyltransferase (CPT) of microsomes that was recently purified as a approximately 54 kDa protein (Murthy and Bieber, Protein Exp. Purif. 3, 75, 1992). First, the N-terminal amino acids of the microsomal CPT showed 100% homology to the sequence described above. Second, during purification of this CPT, the oxidoreductase and the thiol protease activities of the microsomes became separated from the CPT and these other activities were not found in the approximately 900 fold enriched CPT preparations. Third, an antibody to this protein did not immunoprecipitate oxidoreductase of the solubilized microsomal extract but precipitated the CPT. This same protein has been studied by others as the ERp61 (endoplasmic reticulum protein), GRP58 (glucose regulated protein), and HIP-70 (hormone induced protein) but its function was not identified.
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PMID:Carnitine medium/long chain acyltransferase of microsomes seems to be the previously cloned approximately 54 kDa protein of unknown function. 823 44

Estrogen deficiency is associated with bone loss, and estrogen replacement is an effective treatment of this osteoporotic process. This study examines the early (5-120 s) effects of 17 beta-estradiol on the intracellular calcium and phospholipid metabolism in confluent female rat osteoblasts. The cytosolic free Ca2+ concentration ([Ca2+]i) was determined using fura-2/AM as Ca2+ probe. Cells were labeled with myo-[2-3H]inositol or [14C]arachidonic acid for inositol or lipid determination. Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) production were determined by either mass measurement or anion-exchange chromatography or by thin-layer chromatography, respectively. 17 beta-Estradiol (1 pM to 1 nM) increased [Ca2+]i in a biphasic manner within 10 s via Ca2+ influx from the extracellular milieu, as shown by the effects of the calcium chelator EGTA and the Ca2+ channel blockers nifedipine and verapamil, and via Ca2+ mobilization from the endoplasmic reticulum (ER), as shown by the effects of thapsigargin. 17 beta-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP3 and DAG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-(O-carboxymethyl)oxime BSA] and its derivative (O-carboxymethyl)oxime rapidly increased ([Ca2+]i, IP3, and DAG and were full agonists, although they were less potent than the free estradiol. They had the same action time course and acted via Ca2+ influx and Ca2+ mobilization from ER. Tamoxifen, a potent inhibitor of genomic steroid responses, did not block the rapid increase in Ca2+, IP3, and DAG induced by estradiol. Finally, inhibitor of phospholipase C (neomycin) and pertussis toxin abolished the effects of 17 beta-estradiol on IP3 and DAG formation. These results suggest that female rat osteoblasts bear non-genomic unconventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C via a pertussis toxin-sensitive G protein.
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PMID:Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors. 826 28


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