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)

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H-type catalytic subunits, which are inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl-binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl-binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS-7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine-glycan-phosphatidylinositol-anchored G2 forms in vivo. ChE1 also possesses an ethanolamine-glycan-phosphatidylinositol-anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.
...
PMID:cDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus--comparison with cholinesterase 1 and cholinesterase 2 produced in vivo. 987 7

By treatment with phosphatidylinositol-specific phospholipase C (PIPLC), we obtained several candidates of glycosylphosphatidylinositol (GPI)-anchored proteins such as 55, 42, 40, and 30 kDa from bovine erythrocyte membrane, in addition to the well-known GPI-anchored protein acetylcholinesterase. In these proteins, the presence of myo-inositol was confirmed by gas chromatography (GC)-mass spectrometry. Among them, the 42-kDa protein was further analyzed by electrospray-ionization (ESI)-mass spectrometry (MS) after hydrolysis by lysyl endoprotease. By liquid chromatography (LC)-ESI-MS analysis, C-terminal peptides bearing the products of GPI (Ct. GPI-peptides) were effectively detected by combination with in-source collision and multifunctional scanning for the several characteristic fragment ions from the GPI-anchor structure. Existence of microheterogeneity was also observed in the Ct. GPI-peptides from the 42-kDa protein. This result was confirmed by analysis with time-of-flight (TOF)-MS. Furthermore, one of the Ct. GPI-peptides was analyzed in ESI-MS-MS mode. Characteristic fragment ions were effectively detected by collision-induced decay. By the result of MS-MS analysis, this GPI-anchor structure was revealed to contain additional N-acetyl hexosamine. By the above-mentioned method, the C-terminal GPI-anchor structure can be easily identified from the target protein even if its amino acid sequence data are not available.
...
PMID:Identification of a new glycosylphosphatidylinositol-anchored 42-kDa protein and its C-terminal peptides from bovine erythrocytes by gas chromatography-, time-of-flight-, and electrospray-ionization-mass spectrometry. 1004 99

Acoustic neurinomas were sequentially extracted with saline and saline-Triton X-100 buffers. Detergent was required to detach the bulk of acetylcholinesterase (AChE), but butyrylcholinesterase (BuChE) was mostly released with saline buffer. Sedimentation analysis and hydrophobic chromatography revealed that neurinomas contain principally amphiphilic AChE tetramers, dimers and monomers, and hydrophilic BuChE tetramers. The AChE dimers and monomers remained amphiphilic after incubation with phosphatidylinositol-specific phospholipase C (PIPLC), after or without prior treatment with alkaline hydroxylamine, which shows that, in contrast to the meningioma AChE dimers and monomers, the neurinoma isoforms are devoid of glycolipid. Neurinoma AChE reacted with the antibodies HR2 and AE1 raised against AChE from human brain or erythrocyte, whereas BuChE bound to a sheep antiserum.
...
PMID:Characterization of molecular forms of acetyl- and butyrylcholinesterase in human acoustic neurinomas. 1053 May 19

In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G(2) form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.
...
PMID:Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin. 1058 85

In order to know whether the histopathological changes of liver, which accompany muscular dystrophy, affect the synthesis of cholinesterases, the distribution and glycosylation of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) forms in normal (NL) and dystrophic Lama2(dy) mouse liver (DL) were investigated. About half of liver AChE, and 25% of BuChE were released with a saline buffer (fraction S(1)), and the rest with a saline-Brij 96 buffer (S(2)). Abundant light (G(2)(A) and G(1)(A)) AChE (87%) and BuChE (93%) forms, and a few G(4)(H) and G(4)(A) ChE species were identified in liver. The dystrophic syndrome had no effect on solubilization or composition of ChE forms. Most of the light AChE and BuChE species (>95%) were bound by octyl-Sepharose, while most light AChE forms (80%), but not BuChE isoforms (15%), were retained in phenyl-agarose. About half of the AChE dimers lost their amphiphilic anchor with phosphatidylinositol-specific phospholipase C (PIPLC), and the fraction of PIPLC-resistant species increased in DL. AChE T and R transcripts were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) of liver RNA. ChE components of liver, erythrocyte, and plasma were distinguished by their amphiphilic properties and interaction with lectins. The dystrophic syndrome increased the liver content of the light AChE forms with Lens culinaris agglutinin (LCA) reactivity. The abundance of ChE tetramers in plasma and their small amount in liver suggest that after their assembly in liver they are rapidly secreted, while the light species remain associated to hepatic membranes.
...
PMID:Muscular dystrophy alters the processing of light acetylcholinesterase but not butyrylcholinesterase forms in liver of Lama2(dy) mice. 1100 95

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.
...
PMID:Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes. 1267 81

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.
...
PMID:Molecular properties of acetylcholinesterase in mouse spleen. 1508 30

This study analyzed the expression of muscarinic acetylcholine receptors (mAChRs) in the rat cultured skeletal muscle cells and their coupling to G protein, phospholipase C and adenylyl cyclase (AC). Our results showed the presence of a homogeneous population of [(3)H]methyl-quinuclidinyl benzilate-binding sites in the membrane fraction from the rat cultured muscle (K(D) = 0.4 nM, B(max) = 8.9 fmol mg protein(-1)). Specific muscarinic binding sites were also detected in denervated diaphragm muscles from adult rats and in myoblasts isolated from newborn rats. Activation of mAChRs with carbachol induced specific [(35)S]GTPgammaS binding to cultured muscle membranes and potentiated the forskolin-dependent stimulation of AC. These effects were totally inhibited by 0.1-1 microM atropine. In addition, mAChRs were able to stimulate generation of diacylglycerol (DAG) in response to acetylcholine, carbachol or selective mAChR agonist oxotremorine-M. The carbachol-dependent increase in DAG was inhibited in a concentration-dependent manner by mAChR antagonists atropine, pirenzepine and 4-DAMP mustard. Finally, activation of these receptors was correlated with increased synthesis of acetylcholinesterase, via a PKC-dependent pathway. Taken together, these results indicate that expression of mAChRs, coupled to G protein and distinct intracellular signaling systems, is a characteristic of noninnervated skeletal muscle cells and may be responsible for trophic influences of acetylcholine during formation of the neuromuscular synapse.
...
PMID:Developing skeletal muscle cells express functional muscarinic acetylcholine receptors coupled to different intracellular signaling systems. 1604 3

Half of congenital muscular dystrophy cases arise from laminin alpha2 (merosin) deficiency, and merosin-deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean +/- SD 1.42 +/- 0.28 micromol acetylthiocholine/h/mg protein, U/mg) was decreased by approximately 50% in dystrophic thymus (0.77 +/- 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT-PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (G2A, 64%) and monomers (G1A, 19%), as well as hydrophilic tetramers (G4H, 9%) and monomers (G1H, 8%). The dimers consisted of glycosylphosphatidylinositol-anchored H subunits. Western blot assays with anti-AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol-specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.
...
PMID:Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice. 1613 75

The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also alpha(1)-adrenoceptors and beta-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of alpha(1A), alpha(1B), alpha(1D), beta(1), and beta(2)-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, alpha(1)-adrenoceptors to 23%, and beta-adrenoceptors to about 50% of control. Changes were subtype specific, as alpha(1A), alpha(1B), and beta(2)-adrenoceptors, but not alpha(1D)-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.
...
PMID:Adaptation to excess acetylcholine by downregulation of adrenoceptors and muscarinic receptors in lungs of acetylcholinesterase knockout mice. 1780 15


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

---