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)

The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.
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PMID:Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells. 151 21

Porcine brain dolichol kinase activity is effectively solubilized by extracting salt-washed microsomes with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). When the detergent-solubilized activity is chromatographed on Sepharose CL-6B, a low amount of dolichol kinase activity is recovered in the void volume, and a dolichol kinase activator (DKA) is eluted (Ve/Vo = 1.9-2.2) with the bulk of the membrane phospholipids. Although only approximately 20% of the activity applied to the Sepharose CL-6B column is detected in the column fractions, virtually all of the original activity is restored when the Vo fraction is recombined with DKA. Endogenous DKA, isolated from brain microsomes, is heat-stable, is extractable with CHCl3/CH3OH (2:1), and has the chemical and chromatographic properties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, approximately 50% of the stimulatory activity is lost when the PC present in the DKA fraction is degraded by purified phospholipase C from Clostridium perfringens. Also consistent with a phospholipid co-factor requirement, the dolichol kinase activity recovered in the partially phospholipid-depleted fraction (Vo) is markedly stimulated by various molecular species of exogenous purified PC or PE, but not by phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, or sphingomyelin. A comparison of defined molecular species shows that PCs containing oleoyl or linoleoyl groups in the 1 and 2 positions are the most stimulatory, suggesting that the fatty acyl moieties are involved in the enzyme-phospholipid interaction. Kinetic analyses indicate that PC enhances the interaction between dolichol kinase and dolichol, the lipophilic substrate, but does not alter the apparent Km for CTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Separation of brain dolichol kinase from endogenous activating factors: evidence that phospholipid enhances the interaction between enzyme and dolichol. 215 52

Recent studies suggested the presence of specific glucocorticoid binding sites on rat liver microsomal membranes. We report here a new solubilization procedure which allows the physicochemical characterization of the microsomal glucocorticoid binding sites. Solubilization was achieved with 2 mM CHAPS in the presence of 5 mM benzamidine. Binding of [3H]cortisol showed a high affinity (Kd = 5.1 x 10(-9) M) and a limited capacity (0.72 pmol/mg of protein). The binding activity was abolished by elevated temperature and pronase. Competition experiments revealed that natural glucocorticoids and progesterone were highly potent competitors whereas dexamethasone and triamcinolone acetonide did not compete. Chromatography on DEAE Trisacryl and heparin Ultrogel confirmed that the solubilized protein is different from corticosteroid binding globulin and the cytosolic glucocorticoid receptor. Treatment of microsomal fractions with phosphatidyl inositol phospholipase C promoted the release of specific binding activity suggesting a putative glycosylphosphatidyl anchor for this protein. This finding may have interesting implications concerning the mechanism of glucocorticoid hormone action.
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PMID:Occurrence of glucocorticoid binding sites in solubilized microsomes from rat liver. 262 26

The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol.
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PMID:A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. 375 91

Membrane-depleted rat liver nuclei contain diacylglycerol (DAG) kinase showing a specific activity which doubles that of the whole homogenate. In contrast, cytoplasmic and plasma membrane marker enzymes attain a specific activity of 0.4% at the most, when nuclear DAG kinase approaches 4.5% of the total tissue activity. The enzyme shows a Km of 161 and 200 microM for ATP in both nuclei and microsomes whereas the Km for DAG is 75 microM in nuclei and 658 microM in microsomes. Octylglucoside, CHAPS and Triton X-100 behave mainly as inhibitors, while deoxycholate stimulates the enzyme activity in both cellular fractions, increasing specific activity (3.2-fold in nuclei and 29.1-fold in microsomes) and decreasing Km for DAG (39 microM in nuclei and 237 microM in microsomes). Phospholipids and ceramide stimulate the enzyme activity in isolated nuclei, while no effect occurs in the microsomal fraction. At variance, sphingosine behaves as an inhibitor in both cellular fractions. DAG kinase also utilizes endogenous substrates mobilized by Bacillus cereus phospholipase C, which hydrolyses nuclear phosphatidylcholine and phosphatidylethanolamine and by phosphatidylinositol-specific phospholipase C, which hydrolyses nuclear PI and PIP. These data indicate that nuclear DAG can be controlled by converting it into phosphatidic acid by the action of a nuclear enzyme and support the contention that protein kinase C activity can be modulated at the nuclear level by a discrete system involving phospholipase C and DAG kinase that could operate independently from the cytoplasm.
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PMID:Diacylglycerol kinase activity in rat liver nuclei. 794 64

A new strategy has been successfully applied to reconstitute the brain specific serotonin 5-HT1A receptor-G protein-adenylate cyclase complex. A mild method of tissue preparation gave a stable, membrane-bound form of the receptor (SBP) which retained its natural lipid content. Treatment of SBP with serotonin (1 microM) and 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propanesulphonate (CHAPS) (2%) solubilized the ligand-receptor-G protein-ligand complex along with the associated phospholipids and cholesterol. Dialysis of this extract (SBDS) against buffer containing 25% ethylene glycol produced a stable, reconstituted and active preparation (SBDSE) of vesicles which upon centrifugal separation followed by gentle resuspension retained 95-100% [3H] 8-OH-DPAT binding activity as well as 60% [3H] GppNHp binding and adenylate cyclase activities of SBDSE. The reconstituted receptor preparation compared well with the membrane-bound form in displaying a similar value for KD (2.1 nM) and a single affinity state for [3H] 8-OH-DPAT binding (Bmax = 118 fmol/mg). However, in sharp contrast to the membrane-bound receptor which was negatively coupled to adenylate cyclase, agonist treatment of the solubilized and reconstituted receptor resulted in an increase in adenylate cyclase. This change in receptor-adenylate cyclase coupling following reshuffling of membrane lipids during solubilization and reconstitution suggested that membrane lipids could have a profound effect on receptor-effector coupling. To study the effect of membrane lipid composition on receptor-mediated signal transduction in a stabler and more natural system, neural cells derived from different parts of the brain (hippocampus, HN2; CNS, NCB-20; dorsal root ganglion, F-11) and a non-neural cell line (CHO), all with differing membrane lipid compositions, were selected. Since no known cell line contains the serotonin 5-HT1A receptor (5-HT1A-R), stable transfection of the selected cell lines with a DNA construct encoding the human 5-HT1A-R was carried out and this resulted in a late increase of [3H] 8-OH-DPAT binding in the stationary phase only in the cell lines of neural origin. In the non-neural cell line (CHO), which also displayed marked difference in membrane lipids, the receptor was positively coupled to the phospholipase C-IP3-[Ca2+]i cascade. Even though GPLC was present in the NCB-20 and F-11 cells as evidenced by a bradykinin receptor-mediated increase in inositol phosphates in these cells 8-OH-DPAT treatment resulted in no change in phospholipase C in any of the cell lines of neural origin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of lipids in receptor mediated signal transduction. 800 19

The tegument of trematodes serves as a dynamic host-parasite interface where surface antigens are shed in a process of immune evasion. Phospholipases, which could provide an enzymatic mechanism for release of glycosylphosphatidylinositol (GPI)-anchored proteins, were detected in detergent extracts of adult worms of Fasciola hepatica and cercaria and adult worms of Schistosoma mansoni. The enzymatic activities were partially characterized from both adult worm species and demonstrated a preference for [3H]GPI substrate over [3H]PI. Lipase activities from both species were sensitive to sulfhydryl-modifying reagents and the detergents CHAPS and n-octylglucoside. The presence of 1 M ammonium sulfate increased the enzyme activity in adult worms of both species by 8-11-fold and in cercaria by 146-fold, whereas other conditions of high ionic strength were inhibitory. Such stimulation suggested dissociation of a negative inhibitor which is prominent in the cercarial stage. The schistosome extract, which was partially sensitive to cation chelators and o-phenanthroline, contained a GPI-phospholipase D activity. In contrast, the F. hepatica extract contained a cation-independent phospholipase C activity which was partially purified and shown by gel filtration to have a molecular mass of 30,000-80,000.
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PMID:Detection and partial characterization of glycosylphosphatidylinositol-specific phospholipase activities from Fasciola hepatica and Schistosoma mansoni. 839 Jun 13

Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and (alpha-mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X-114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X-114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosylation. The use of a Con-A binding assay demonstrated that both the enzymes are glycosylated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space.
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PMID:Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases. 989 Jul 47

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy.
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PMID:Phosphatidylinositol synthesis in mycobacteria. 998 74

IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.
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PMID:Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi. 1556 2


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