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

---

Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.
...
PMID:Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. 167 99

Dimeric acetylcholinesterase is anchored in the cell membrane by a glycosyl-phosphatidylinositol attached to the C-terminus of the protein. The complex glycan contains an antigenic epitope, the cross-reacting determinant (CRD), which is only revealed after removal of the diradylglycerol by phosphatidylinositol-specific phospholipase C (PI-PLC) but is cryptic in the amphiphilic form. Polyclonal antibodies were raised against the CRD of vertebrate acetylcholinesterase. The purified anti-CRD antibodies recognized only the PI-PLC treated hydrophilic forms of acetylcholinesterase from bovine erythrocytes and Torpedo, and of variant surface glycoprotein from trypanosomes but not the corresponding amphiphilic proteins. Competition experiments showed that inositol-1,2-cyclic phosphate and glucosamine inhibited the binding of the antibodies to the CRD. Furthermore, binding of the anti-CRD antibodies to acetylcholinesterase containing N-methylated glucosamine was markedly reduced. The amphiphilic N-methylated enzyme is less sensitive to digestion with PI-PLC than the non-methylated form. From our results we conclude that inositol-1,2-cyclic phosphate and glucosamine, especially the free amine group of this residue, contribute significantly to the epitope recognized by the anti-CRD antibodies.
...
PMID:Production and characterization of antibodies against the cross-reacting determinant of glycosyl-phosphatidylinositol-anchored acetylcholinesterase. 169 31

Several proteins including bovine erythrocyte acetylcholinesterase are anchored in the membrane through glycoinositol phospholipids containing an alkyl linkage at the sn-1 position of the glycerol. However, the existence of 1-alkyl-2-acyl-sn-glycero-3-phosphoinositol (alkylacyl-GPI) in biological systems has not been demonstrated. In this study, we identified the presence of alkylacyl-GPI in bovine erythrocytes by the following criteria: (1) TLC-Rf value, (2) radyllyso-GPI was produced after phospholipase A2 treatment of the diradyl-GPI, and (3) benzoate derivatives of alkylacylglycerols produced by phospholipase C hydrolysis of diradyl-GPI had the same retention time as that of authentic alkylacylglycerobenzoates on normal-phase HPLC. Diradyl-GPI consisted of 5-10% alkylacyl-GPI. Reverse-phase HPLC analysis of alkylacylglycerobenzoates derived from bovine erythrocyte alkylacyl-GPI showed a multiplicity of species with 18:0-20:4 (11.7%), 16:0-18:1 + 18:0-18:2 (34.9%), and 18:0-18:1 (19.4%) being the major components. Composition of alkyl chains of alkylacyl-GPI from bovine erythrocytes was similar to the reported value for alkylacylglycerols isolated from the glycoinositol phospholipid anchor of bovine erythrocyte acetylcholinesterase. Based on these results, we suggest that alkylacyl-GPI serves as a precursor for the glycoinositol phospholipid of the anchored proteins.
...
PMID:Occurrence of ether-containing inositol phospholipids in bovine erythrocytes. 182 38

We have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of bacterial origin. Exposure to PtdIns-PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns-PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns-PLC, in the presence of the protein-synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns-PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti-AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns-PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns-PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dimyristoyl-sn-glycerol were able to increase AChE activity by 35-40% at concentrations of 25 micrograms/ml. A higher concentration of 1,2-dioctanoyl-sn-glycerol (70 micrograms/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol-12-myristate-13-acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns-PLC. Our results suggest the involvement of glycophosphatidyl membrane-anchor breakdown products as putative second messengers in the parasite S. mansoni.
...
PMID:Phosphatidylinositol-specific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni. 184 73

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.
...
PMID:Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes. 184 54

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.
...
PMID:Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases. 184 55

Using phosphatidylinositol-glycan (PtdIns-glycan) anchored acetylcholinesterase from bovine erythrocytes as substrate, we found PtdIns-glycan-anchor-degrading activity in rat liver and serum [corrected]. The hepatic enzyme was only soluble in detergents, whereas the serum enzyme occurs as soluble, slightly amphiphilic protein. Using 3-trifluoromethyl-3-(m- [125I]iodophenyl)diazirine-labelled acetylcholinesterase as substrate, we showed that the hepatic anchor-degrading enzyme had a cleavage specificity of a phospholipase C, whereas the serum enzyme was a phospholipase D. Both enzyme exhibited maximal activity in slightly acidic conditions and at low ionic strength. They had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 0.1 microM and 0.16 microM, respectively). Both hepatic PtdIns-glycan-specific phospholipase C and serum PtdIns-glycan-specific phospholipase D gave a large increase in activity between 0.1-10 microM Ca2+, indicating that PtdIns-glycan-specific phospholipases are only marginally active at physiological intracellular Ca2+ concentrations. The enzymes were inhibited by heavy metal chelating agents such as 1,10-phenanthroline and 2,2'-bipyridyl but not by the corresponding Fe2+ complexes or non-chelating analogues, indicating that they both require a heavy metal ion for the expression of catalytic activity in addition to Ca2+. Another interesting property of PtdIns-glycan-specific phospholipases is their inactivation by bicarbonate and cyanate. The inactivation was time- and pH-dependent and could be reversed by dialysis. These observations are in agreement with a covalent modification of the enzymes by carbamoylation.
...
PMID:Enzymatic properties of phosphatidylinositol-glycan-specific phospholipase C from rat liver and phosphatidylinositol-glycan-specific phospholipase D from rat serum. 184 23

The kinetic analysis of Apis mellifera acetylcholinesterase inhibition by the carbamate pirimicarb showed that native and detergent-solubilized membrane enzyme exhibited slightly different carbamylation kinetics. The acetylcholinesterase form sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) was carbamylated more rapidly (kapp = 36.4 X 10(-3) min-1) than the PI-PLC-resistant counterpart (kapp = 10.13 X 10(-3) min-1) which had a behavior close to that of the soluble tryptic enzyme (kapp = 11.89 X 10(-3) min-1). A difference in acetylcholinesterase sensitivity towards pirimicarb was also observed between foraging and emerging bees. These results show that the molecular structure, the mode of preparation and the source of acetylcholinesterase from the bee head should be taken into account in accurate toxicological studies.
...
PMID:Differential response of Apis mellifera acetylcholinesterase towards pirimicarb. 191 59

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.
...
PMID:Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase. 203 16

In the culture supernatant of Cytophaga sp. we detected an enzyme that converted glycosylphosphatidyl-inositol-anchored acetylcholinesterase to the hydrophilic form. This enzyme had a cleavage specificity of a phospholipase C. It hydrolyzed phosphatidylinositol but did not act on phosphatidylcholine. On gel filtration the enzyme migrated with an apparent molecular mass of about 17 kDa. It displayed maximal activity between pH 6-6.5 and did not require cofactors for the expression of catalytic activity. Mercurials and zinc ions inhibited the enzyme and its activity also decreased with increasing ionic strength in the assay. With acetylcholinesterase as substrate optimal activity was obtained in pure micelles of Triton X-100, whereas in mixed micelles containing Triton X-100 and phosphatidylcholine the activity was reduced. The enzyme from Cytophaga sp. showed little activity towards acetylcholinesterase embedded in intact membranes where more than 1000-times higher concentrations of phosphatidylinositol-specific phospholipase C was necessary to solubilize acetylcholinesterase as compared to acetylcholinesterase in detergent micelles.
...
PMID:Cholinesterase solubilizing factor from Cytophaga sp. is a phosphatidylinositol-specific phospholipase C. 204 78


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---