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)

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes. The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens. Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin. Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis. The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.
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PMID:Increase in osmotic fragility of bovine erythrocytes induced by bacterial phospholipases C. 630 97

The mode of acetylcholinesterase release from mammalian erythrocyte membranes by the action of phosphatidylinositol(PI)-specific phospholipase C of Bacillus thuringiensis was studied. As regards intact erythrocytes, a larger amount of acetylcholinesterase was released from sheep or bovine erythrocytes than from horse erythrocytes. From horse erythrocyte ghosts, acetylcholinesterase was more easily released than from intact cells. Bovine erythrocyte acetylcholinesterase released by PI-specific phospholipase C was purified by column chromatography on DEAE-cellulose, affinity gel and Sepharose 6B, to a homogeneous state, as indicated by polyacrylamide gel electrophoresis, with a recovery of 39%. Also, bovine erythrocyte acetylcholinesterase was solubilized by Triton X-100 and partially purified. The properties of these acetylcholinesterase preparations obtained by the action of PI-specific phospholipase C and/or Triton X-100 were studied in detail. On elution from the Sepharose 6B column, Triton X-100-solubilized acetylcholinesterase was eluted at the void volume while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the region corresponding to M.W. 250,000. Furthermore, the heat stability of acetylcholinesterase purified after solubilization with PI-specific phospholipase C was higher than that of the Triton X-100-solubilized acetylcholinesterase. The close association and direct interaction of PI with acetylcholinesterase in the erythrocyte membrane was suggested by the above results.
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PMID:Acetylcholinesterase release from mammalian erythrocytes by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis and characterization of the released enzyme. 650 Dec 51

A fluorescent acyldicholine, bis-(choline)N-[4-nitrobenzo-2-oxa-1,3-diazol-7-yl]-iminodiprop ionate (BCNI), was synthesized and its capacity to associate with acetylcholinesterase and the nicotinic acetylcholine receptor examined. The fluorescent bisquaternary diester competitively inhibits acetylcholinesterase with a Ki of 0.46 microM. Binding is accompanied by a large decrease in BCNI fluorescence and a 40% reduction in enzyme tryptophanyl fluorescence due to spectral overlap between BCNI absorption and the fluorescence emission of tryptophanyl residues on the enzyme. BCNI titrations show a stoichiometry of one site per subunit and a dissociation constant of 0.2 microM. BCNI also inhibits the initial rate of alpha-toxin binding to the membrane-associated nicotinic acetylcholine receptor and yields a protection constant (Kp) of 0.26 microM. Prior exposure of BCNI to the receptor increases the affinity of the complex, and after equilibration Kp is found to be 0.11 microM. Fluorescence titrations reveal that BCNI binds with 1:1 stoichiometry to alpha-toxin sites on the receptor with a dissociation constant of 0.22 microM. Agonists and antagonists, but not local anesthetics, compete with BCNI binding. BCNI behaves as a competitive antagonist on receptors from the snake neuromuscular junction and from BC3H-1 cells. The 4-nitrobenzo-2-oxa-1,3-diazole fluorophore in BCNI shows a hypsochromatic shift and an enhancement of quantum yield when bound to the receptor but is quenched when associated with acetylcholinesterase. Thus, despite the similarity in dissociation constants, the fluorophore exists in very different environments when bound to the two proteins.
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PMID:Interaction of a fluorescent acyldicholine with the nicotinic acetylcholine receptor and acetylcholinesterase. 654 67

A dimeric form of acetylcholinesterase from the electric organ of Torpedo californica was solubilized by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The solubilized enzyme had a sedimentation coefficient of 7.3S which was not modified by detergents. The high salt-soluble asymmetric forms of acetylcholinesterase were not solubilized by the phospholipase. Our data suggest that the hydrophobic dimer of acetylcholinesterase may be associated with the plasma membrane through a specific interaction involving phosphatidylinositol.
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PMID:A hydrophobic dimer of acetylcholinesterase from Torpedo californica electric organ is solubilized by phosphatidylinositol-specific phospholipase C. 663 70

The role of phosphatidylinositol-specific phospholipase C (PIase C) in a) the enigmatic phosphatidylinositol (PI) turnover and b) in our understanding of membrane enzyme-PI interactions is the subject matter of this article. PIase C is present in both procaryotes and eukaryotes. This enzyme is considered to be involved in the cells PI breakdown which occurs in response to several external stimuli. Recent information on the physical properties, Ca2+ requirement, cellular localization and modulation of the activity of PIase C of mammalian systems can help to evaluate the PI turnover from a new angle. Existing evidence suggests that Ca2+-dependent PI breakdown is probably mediated through the cytosolic and particulate PIase C while a Ca2+ independent pathway is catalyzed by a lysosomal enzyme. Apparently PI turnover may be operating through more than one mechanism. The association of this phenomenon with a membrane receptor event linked with "Ca2+ gating" may have to be reconsidered. Modulation of the PIase C activity by unsaturated amphiphiles or the presence of this enzyme in different physico-chemical forms could be a potential regulatory feature. Hydrolysis of membrane PI of a number of cells and tissues by the bacterial PIase C has been shown to cause substantial release of acetylcholinesterase, alkaline phosphatase and 5'-nucleotidase in free, soluble form. Other membrane enzymes, e.g., alkaline phosphodiesterase I, L-leucyl-beta naphthyl amidase and Ca2+ or Mg2+ ATPase are not affected. These results indicate a specific interaction between PI and certain enzymes in membranes. The chemical nature of this linkage, whether it is covalent or non-covalent, has also been explored and has provided intriguing insight into this phenomenon. New findings also indicate that hydrolysis of PI by PIase C also can cause modifications in membrane-enzyme activities, e.g., adenylate cyclase.
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PMID:Minireview. Phosphatidylinositol specific phospholipases C. 708 67

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (less than 5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.
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PMID:Electrokinetic behavior of inside-out vesicles from human red cell membranes. 711 10

Amphiphilic monomers and dimers of acetylcholinesterase (AChE) and hydrophilic tetramers of butyrylcholinesterase (BuChE) were released by extracting human meningioma with Tris-saline and Tris-saline-Triton X-100 buffers. The amphiphilic or hydrophilic behavior of the AChE and BuChE forms was assessed by sedimentation analysis, hydrophobic chromatography and Triton X-114 phase-partitioning. A significant fraction of the amphiphilic AChE species was converted into hydrophilic components by incubation of the soluble enzyme with phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, this fraction being increased by a double treatment with PIPLC and alkaline hydroxylamine. A significant amount of the membrane-bound AChE was released by incubation with PIPLC. These results demonstrate that AChE forms in meningioma are attached to the membrane via glycosylphosphatidylinositol, although part of the enzyme forms are resistant to PIPLC.
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PMID:Monomers and dimers of acetylcholinesterase in human meningioma are anchored to the membrane by glycosylphosphatidylinositol. 747 60

This study reports, for the first time, the detection of glycosylphosphatidylinositol (GPI) membrane anchors in proteins of a pathogenic fungus, Paracoccidioides brasiliensis. Taking into account that fungal antigens are found in the sera of paracoccidioidiomycosis patients and that cleavage of this glycolipid by phospholipases is a means of selective protein release, the presence of an enzyme with this property has also been investigated. Using a methodological approach in which the proteins were immobilized on nitrocellulose, treated with phospholipase C of Trypanosoma brucei and then probed with antibodies which recognize the 1,2-cyclic-phosphate inositol moiety formed as a reaction product in proteins bearing the glycolipid anchor, it was possible to detect a major glycoprotein in the 80- to 90-kDa range, as well as two other minor species of 66 and 43 kDa. All of them bind to Concanavalin-A and are also substrates of a very potent fungal phospholipase C which is inhibited by p-chloromercuri-phenylsulfonic acid and is insensitive to EDTA. The integrity of glycosylphosphatidylinositol anchors in proteins of P. brasiliensis is impaired by 0.1 M NaOH, a finding indicative of a diacyl glycerolipid moiety which is quite surprising since it is, with the exception of African trypanosomes surface proteins and Torpedo acetylcholinesterase, an uncommon feature among GPIs in general. The present findings may have implications in the pathology of paracoccidiodomycosis.
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PMID:Paracoccidioides brasiliensis expresses both glycosylphosphatidylinositol-anchored proteins and a potent phospholipase C. 761 72

The presence of GPI anchors and phospholipases capable of solubilizing them in Trypanosoma cruzi has been investigated in epimastigotes, metacyclic trypomastigotes from axenic cultures and tissue culture trypomastigotes. The GPI anchored proteins in epimastigote forms are scarce when compared to their abundance in the parasite forms which can infect mammals, and GPI-solubilizing phospholipases C have been found in all life cycles stages. In epimastigote and metacyclic forms, the activity is found in the soluble fraction upon cell lysis, whereas in tissue cultured trypomastigotes it is membrane bound and, being mostly sensitive to p-chloromercuriphenylsulfonate, resembles closely the GPI specific phospholipase of Trypanosoma brucei. Sequential immunoprecipitations with monoclonal antibodies and anti-CRD indicated the presence of several sub-populations among the surface proteins of metacyclic trypomastigotes, five of these belonging to the GPI-anchored 90 kD family. Among this family, the epitopes recognized by MAb-1G7 are present in three members, one of them also expressing the 3F6 epitope. There are 2 members recognized only by MAb-3F6 but not by MAb-1G7, one of them being probably galactosylated on the GPI since it can be immunoprecipitated by anti-CRD. Very strangely, the epitope recognized by the MAb-WIC29.26 was always present on the gp72, as originally described, but under certain circumstances appeared cryptic on one of the 90 kD species. During epimastigote transformation into metacyclic trypomastigotes in vitro, the ability of the GPI of the 1G7-antigen to be solubilized by phospholipase C and D varies depending on the age of the culture and presence or absence of fetal calf serum. Different patterns of solubilization were also obtained for 1G7-Ag, depending on whether the test is performed with parasite lysates or with antigen affinity purified from them. Our data indicate that the phospholipase C resistance observed does not arise from acylation on the inositol, as previously described for acetylcholinesterase from human erythrocytes, being rather due to factors which either modify the GPI or affect the action of the phospholipases. Previously unreported resistance to glycosylphosphatidylinositol-specific phospholipase D has been observed both to glycosylphosphatidylinositol-specific phospholipase D has been observed both to 1G7-Ag and 10D8-Ag, the GPI-anchored mucynlike protein which is acceptor of sialic acid in metacyclic forms. Our findings are discussed in the light of the presently known structures of GPI in this parasite, and imaginative speculation on biological roles for the GPI phospholipase system in T. cruzi is also provided.
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PMID:Proteins anchored via glycosylphosphatidylinositol and solubilizing phospholipases in Trypanosoma cruzi. 767 May 41

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


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