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)

A 247 000 x g particulate fraction from a moderately halophilic halotolerant bacterium incorporated [14C] glucose added as UDP[14C]glucose and 32P-labeled phosphatidylglycerol into glucosylphosphatidylglycerol. Exogenously added phosphatidylglycerol was available to the enzyme only when dispersed in a detergent, preferably Triton X-405, by sonication. The 14C- or 32P-labeled glucosylphosphatidylglycerol was degraded with phospholipase C. The water soluble product formed was isolated and identified by paper chromatography as glucosylglycerolphosphate. The system required Mg2+ or Ca2+ for activity. KCl and NaCl were inhibitory even when added at low concentrations.
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PMID:Glycolipids of a halotolerant, moderately halophilic bacterium. 69 36

Glycosyl phosphoinositol (GPI) anchors on proteins can be modified by palmitoylation of their inositol residue, which makes such anchors resistant to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) (Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T.L. (1988) J. Biol. Chem. 263, 18766-18775). Mannosylated GPI lipids made in trypanosomal and mammalian cells can also be inositol-acylated, indicating that inositol acylation may be a normal step in GPI anchor synthesis. We find that Saccharomyces cerevisiae mutants blocked in dolichyl phosphate mannose synthesis accumulate a lipid that can be radiolabeled in vivo with [3H]myo-inositol, [3H]GlcN, and [3H]palmitic acid. This lipid is resistant to PI-PLC, yet sensitive to mild alkaline hydrolysis, and has been characterized as GlcN-phosphatidylinositol (PI), fatty acylated on its inositol residue. When yeast membranes are incubated with UDP-[14C] GlcNAc, 14C-labeled GlcNAc-PI and GlcN-PI are made. Addition of ATP and CoA, or of palmitoyl-CoA to incubations results in the synthesis of [14C]GlcN-(acyl-inositol)PI. This lipid is also made when membranes are incubated with [1-14C]palmitoyl-CoA and UDP-GlcNAc. We propose that acyl CoA is the donor in inositol acylation of GlcN-PI, and that GlcN-(acyl-inositol)PI is an obligatory intermediate in GPI synthesis.
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PMID:Inositol acylation of a potential glycosyl phosphoinositol anchor precursor from yeast requires acyl coenzyme A. 131 31

Chronic injections of epidermal growth factor (EGF) or the beta-adrenergic receptor agonist isoprenaline resulted in rat parotid gland hypertrophy and hyperplasia. Introduction of a polyclonal antibody to EGF or the EGF-receptor (EGF-R) caused a specific retardation of acinar cell proliferation when injected along with the growth factor. Meanwhile, only the antibody to EGF-R caused a dose-dependent retardation of proliferation on co-administration with isoprenaline both in vivo and in vitro. The antibody injected alone had no effect on cell growth. When cells were incubated in the presence of EGF, plasma membranes from isoprenaline-treated and control animals showed phosphorylation of the EGF-R tyrosine moieties and transient increases in membrane-associated phospholipase C gamma. Isoprenaline did not stimulate phosphorylation of the EGF-R in isolated plasma membranes. However, activation of the phosphotyrosine-signalling pathway could be duplicated by incubating isoprenaline-treated acinar cells, but not control cells, with bovine galactosyltransferase. Immunopurified EGF-R demonstrated variations in reactivity with two different lectins after treatment of the cells with the beta-agonist as well as increased galactosyltransferase substrate capacity in vitro. In addition, incubation of intact acinar cells and isolated plasma-membrane fractions from isoprenaline-treated rats with UDP-[14C]galactose resulted in an increased incorporation of label into the EGF-R. The results suggest that the carbohydrate moiety of the EGF-R has been altered in isoprenaline-treated animals allowing galactosyltransferase now to recognize this receptor. This interaction may in part mediate proliferation of parotid acinar cells. Indeed, we have previously shown that an antibody to galactosyltransferase is capable of blocking isoprenaline-mediated acinar cell proliferation in vivo [Humphreys-Beher, Schneyer, Kidd & Marchase (1987) J. Biol. Chem. 262, 11706-11713].
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PMID:A novel mechanism for isoprenaline-stimulated proliferation of rat parotid acinar cells involving the epidermal growth factor receptor and cell surface galactosyltransferase. 162 94

Decay-accelerating factor (DAF) is anchored in cell membranes by a glycosyl-plasmanylinositol (GPI) moiety that is transferred to it en bloc in the rough endoplasmic reticulum. To analyze the biochemical reactions involved in preassembly of this structure, a human hematopoietic cell-free system was employed. Incubation of cell extracts with UDP-[3H]GlcNAc and butanol partitioning of reaction mixtures yielded two products similar in TLC mobility to intermediates described in Trypanosoma brucei. Both species were sensitive to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C, indicative of association of [3H]GlcNAc label with a plasmanylinositol-containing acceptor. In contrast to trypanosome intermediates, which contain phosphatidylinositol (1,2-diacylglycerophosphoinositol), however, alkali treatment and phospholipase A2 digestion generated butanol-phase products characteristic of glycosylated plasmanylinositol (1-alkyl-2-acylglycerophosphoinositol). Kinetic and pulse-chase experiments indicated that the slower-migrating species was a product of the faster and that it, but not the faster, was sensitive to both GPI-specific phospholipase D and nitrous acid deamination, consistent with conversion of GlcNAc- to GlcN-plasmanylinositol. Accordingly, acetic anhydride acetylation retransformed the slower species back to the faster. Further incubation with cell extracts converted the slower species into more polar products. Lysates of normal and of affected blood leukocytes from two paroxysmal nocturnal hemoglobinuria (PNH) patients supported assembly of the two intermediates within 1 min. Thus, the initial enzymes mediating human GPI-anchor assembly are GlcNAc-plasmanylinositol transferase and GlcNAc-plasmanylinositol deacetylase, their substrates contain plasmanylinositols, and the products of their activities are normal in affected PNH cells.
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PMID:Assembly and deacetylation of N-acetylglucosaminyl-plasmanylinositol in normal and affected paroxysmal nocturnal hemoglobinuria cells. 170 86

The glycosyl phosphatidylinositol (GPI) anchors that attach certain proteins to membranes are preassembled by sequential addition of glycan components to phosphatidylinositol (PI) before being transferred to nascent polypeptide. A cell-free system consisting of trypanosome membranes has been reported to catalyze GPI biosynthesis (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800; Menon, A. K., Schwarz, R. T., Mayor, S., and Cross, G. A. M. (1990) J. Biol. Chem. 265, 9033-9042). We now describe conditions for studying the initial steps of GPI biosynthesis in extracts of murine lymphoma cells. Two chloroform-soluble products, tentatively identified as [6-3H]GlcNAc-PI and [6-3H]GlcN-PI were generated during incubations of EL4 cell lysates with UDP-[6-3H]GlcNAc. The involvement of PI in the reaction was established by the sensitivity of the products to hydrolysis by PI-specific phospholipase C and the finding that the addition of exogenous PI to the incubation stimulated the reaction. The minor, more polar product was sensitive to nitrous acid cleavage and was converted to the major product, as judged by TLC, after treatment with acetic anhydride. The glycolipids generated in lymphoma extracts appeared to be the same as the products produced in parallel incubations with trypanosome membranes. Analysis of available lymphoma mutants deficient in Thy-1 surface expression revealed that extracts of the class A, C, and H mutants are completely defective in synthesizing GlcNAc-PI and GlcN-PI.
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PMID:Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants. 182 68

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.
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PMID:Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities. 190 57

Extracellular ATP and UTP produced a rapid accumulation of inositol phosphates in human airway epithelial cells (CF/T43). The order of agonist potencies for a series of nucleotide analogues differed markedly from that of the classically described P2x- or P2y-purinergic receptors. UTP was the most potent agonist and was fully efficacious; ATP and adenosine-5'-O-(3-thiotriphosphate) were also full agonists. In contrast, 2-methylthio-ATP, adenosine-5'-O-(2-thiodiphosphate) and alpha,beta-methylene-ATP were without effect. ADP and UDP had little or no effect at concentrations as high as 100 microM, and deoxyribose and dideoxyribose compounds were inactive. The effects of ATP and UTP were not additive, whereas bradykinin- or histamine-stimulated inositol phosphate production was additive with the effects of ATP or UTP. Preincubation of cells with either UTP or ATP resulted in a parallel loss of responsiveness to both agonists. Desensitization was specific for the response to nucleotides, because no ATP- or UTP-induced effect on the response to histamine or bradykinin was observed. Pertussis toxin treatment of CF/T43 cells produced a 30-40% decrease in the response to ATP or UTP, which correlated with the ADP-ribosylation of 41- and 43-kDa proteins. Bradykinin and histamine responses were not modified by pertussis toxin. Guanine nucleotides had little effect on the inositol phosphate response in intact CF/T43 cells at concentrations below 100 microM. However, in streptolysin-O-permeabilized cells GTP-gamma S produced a concentration-dependence activation of inositol phosphate formation. UTP or ATP had little effect in permeabilized cells in the absence of guanine nucleotides but markedly increased inositol phosphate formation in the presence of guanine nucleotides. Taken together, these results suggest that UTP and ATP activate a 5'-nucleotide receptor on CF/T43 cells that is distinct from the classically defined P2x- and P2y-purinergic receptors. Activation of phospholipase C by this receptor involves, at least in part, a guanine nucleotide-binding regulatory protein.
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PMID:Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells. 194 36

In human skin fibroblasts, low concentrations of extracellular ATP stimulated 45Ca2+ efflux from a slow-turnover intracellular pool, accompanied by inositol phosphate generation. These effects of ATP were not due to a generalized increase in plasma-membrane permeability. The EC50 (concn. giving 50% stimulation) for ATP was dependent on Ca2+ and Mg2+ concentrations in a manner which indicates that a form of ATP uncomplexed with bivalent cations is the active species. The rank order of potency of nucleotides was: ATP = UTP greater than adenosine 5'-[gamma-thio]triphosphate greater than ITP greater than ADP greater than UDP greater than other nucleoside triphosphates. Adenosine 5'-[alpha beta-methylene]triphosphate, adenosine 5'-[beta gamma-methylene]triphosphate and 2-methylthio-ATP were inactive. Thus the nucleotide specificity of this receptor is different from that of previously characterized P2 purinoceptors. Nucleotide-stimulated 45Ca2+ mobilization and inositol phosphate production were markedly inhibited by phorbol ester, and partially inhibited by pertussis-toxin pretreatment. These findings suggest that the coupling of nucleotide receptor to phospholipase C is mediated both by a pertussis-toxin-sensitive G-protein and by a pertussis-toxin-insensitive mechanism.
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PMID:Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts. 259 9

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.
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PMID:Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase. 295 69

Bilirubin UDP-glucuronyltransferase displays marked latency in native microsomes. To examine whether this latency correlates with structural integrity of the microsomal vesicles and reflects lumenal orientation of the enzyme's catalytic center, we analyzed the relationship between transferase activity and the degree of expression of mannose (Man)-6-phosphatase, which is a marker enzyme of the cisternal face of the ER membrane. Using detergent, sonication, or the pore-forming Staphylococcus aureus alpha-toxin to breach the microsomal membrane permeability barrier, we found that after each of these pretreatments a remarkably close direct relationship existed between latency changes for bilirubin UDP-glucuronyltransferase and Man-6-phosphatase. This finding suggested that the transferase may have the same transverse topology as the phosphohydrolase. We also compared the effects of membrane-impermeant proteinases on bilirubin UDP-glucuronyltransferase activity in native and disrupted microsomes. Whereas the unspecific proteinase nagarse markedly inactivated (to less than 30% of activities in controls) the transferase in disrupted microsomes, treatment with the proteinase had little effect on transferase activity in sealed microsomal vesicles. The results suggest that the active site of bilirubin UDP-glucuronyltransferase is on the lumenal face of the endoplasmic reticulum membrane. It was also found that activation of transferase activity by UDP N-acetylglucosamine, which is the presumed allosteric effector of UDP-glucuronyltransferase, was markedly altered by relatively small changes in structural integrity of the microsomes and totally abolished when latency of Man-6-P hydrolysis fell below approximately 80%. Collectively, these findings demonstrate that the microsomal membrane permeability barrier is a major determinant of expression of microsomal UDP-glucuronyltransferase activity and that quantitative assessment of integrity of the microsomes is essential for studying kinetic properties and regulation of microsomal UDP-glucuronyltransferase.
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PMID:Topology and regulation of bilirubin UDP-glucuronyltransferase in sealed native microsomes from rat liver. 313 Aug 1


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