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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)

Plasma membrane preparations from KA31 (mouse) cells contained receptors for the binding of Rauscher murine leukemia virus (R-MuLV) envelope glycoprotein, gp70. This binding was demonstrated by gel filtration of a mixture of the microsomal fraction of the cells and 125I-labeled gp70. A rapid and convenient assay was developed to measure the complex formation between the membrane receptors and gp70 involving specific precipitation of the complex by 3 to 4% polyethylene glycol. The complex formation was responsive to the concentrations of both the receptor and gp70 and also to changes in temperature and pH. The gp70 binding was a noncooperative, saturable process, and an association constant of 3.5 X 10(8) M-1 was estimated from the binding data. The complex formation was reversible and a near-total exchange of 125I-labeled gp70 in the complex was achieved by incubation with excess of unlabeled gp70. The complex formation was inhibited by protein denaturing agents, guanidine-hydrochloride and urea. Pretreatment of the membrane fractions with either chymotrypsin or phospholipase C led to a loss of the membrane-associated receptor activity, indicating that a lipoprotein structure was important for the receptor function, consistent with the observation that nonionic detergents strongly inhibited the complex formation.
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PMID:Characterization of Rauscher murine leukemia virus envelope glycoprotein receptor in membranes from murine fibroblasts. 3 3

The presence of multiple forms of phosphoinositide-specific phospholipase C (PLC), an important enzyme in the cell signal transduction, suggests that specialized functions of tissues and cells may require different modes of PLC regulation. In the present study, we have purified a 54-kDa heparin-binding protein from a 4 M guanidine hydrochloride extract of porcine kidney, and identified it as one of isoenzymes of PLC on the basis of its partial amino acid sequence. Among 194 determined sequences of the porcine protein, 186 residues were identical with those deduced from nucleotide sequence of the cDNA encoding rat PLC-alpha. The subcellular distribution of porcine renal PLC-alpha was examined by Western blotting by using a specific antibody against the purified protein. Quantitation of the Western blots revealed that 70% of PLC-alpha was membrane-associated. Immunohistochemical studies showed a specific localization of PLC-alpha in epithelial cells of distal tubules and collecting ducts of normal porcine kidney, but not in other cells composing the nephron. Moreover, the highest expression of PLC-alpha was observed in apical membranes in these epithelial cells. Thus, this form of PLC is considered to have a specific role in the signal transduction process related to regional renal tubular functions.
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PMID:Localization of phosphoinositide-specific phospholipase C-alpha in porcine kidney. 133 48

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.
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PMID:Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells. 153 35

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.
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PMID:Modification of phospholipase C and phospholipase A2 activities during poliovirus infection. 255 46

The association of several phosphoproteins with the synaptosomal plasma membrane (SPM) was investigated by phosphorylating SPM fractions from neonatal rat brain in the presence of Ca2+ and then exposing these to a variety of agents. Extraction of the major acidic phosphoproteins, GAP-43, pp40, and pp80, was assessed by two-dimensional gel electrophoresis and fluorography. All three proteins were best extracted from the membrane by high pH and by guanidine hydrochloride. GAP-43 was not extracted in the presence of either low- or high-ionic-strength buffers, reducing agents, or chelating agents; pp80 and pp40, however, showed a significant extraction even under low-ionic-strength conditions. Partition experiments with Triton X-114 revealed an amphiphilic behavior for GAP-43 and a strong affinity for hydrophobic environments for pp80 and pp40. None of the phosphoproteins was released from the membrane by the use of a phosphatidylinositol-specific phospholipase C. The extraction properties of GAP-43, pp80, and pp40 are similar to those of known extrinsic membrane proteins and therefore suggest that these phosphoproteins are peripheral rather than integral to the membrane compartment.
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PMID:Extraction of major acidic Ca2+ dependent phosphoproteins from synaptic membranes. 322 71

Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [14C]HCHO and NaCNBH3, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH3CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH3CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH3CN. CH3CN was removed in a mixture of constant composition (approximately 11% H2O-89% CH3CN), so that a final CH3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A215 and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.
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PMID:Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase. 771 Jan 3

The extracellular phospholipase D (PLD) gene from Streptomyces antibioticus was cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form in E. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCl and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S. antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus and Streptomyces sp., and contained a conserved region with S. chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region XPLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.
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PMID:Phospholipase D from Streptomyces antibioticus: cloning, sequencing, expression, and relationship to other phospholipases. 776 69

A role for phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as a mechanism of alpha 2-adrenergic signal transduction in rabbit tracheal epithelial cells (tracheocytes) was investigated in isolated cells grown in in vitro culture and prelabeled with myo-[3H]inositol (3 microCi/ml) for 72 h. Breakdown of polyphosphoinositides was measured by using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate [PtdIns(4)P], and PtdIns(4,5)P2. Inositol phosphates were separated by ion-exchange column chromatography. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agonists clonidine and 1-(2,6-dichlorobenzylideneamino)guanidine (guanabenz) produced a rapid transient accumulation of inositol trisphosphate and inositol 4,5-bisphosphate and breakdown of [PtdIns(4)P] and PtdIns(4,5)P2. The alpha 2-adrenergic effects were not blocked by the beta-adrenergic antagonist DL-propranolol or by the alpha 1-adrenergic antagonists prazosin and methylurapidil but were inhibited by pertussis toxin and blocked by yohimbine, an alpha 2-adrenergic antagonist. The 50% effective concentration for guanabenz-stimulated inositol trisphosphate generation was right shifted from 0.3 to 0.9 microM by yohimbine. The results provide the first demonstration of alpha 2A-adrenergic activation of pertussis toxin-sensitive PtdIns(4,5)P2-dependent phospholipase C in mammalian tracheocytes. The findings are consistent with previous observations on alpha 2A-adrenergic-mediated activation of NaCl cotransport in these cells.
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PMID:Activation of PtdIns(4,5)P2-sensitive phospholipase C in rabbit tracheal epithelial cells. 790 69

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1 30.4 microM and the Vmax 1.4 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP.
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PMID:Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes. 861 41

A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
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PMID:Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes. 877 Nov 95


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