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Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C.

ABSTRACT: Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.

SUBMITTER: Futerman AH

PROVIDER: S-EPMC1144721 | biostudies-other | 1985 Mar

REPOSITORIES: biostudies-other

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Structure of the glycosyl-phosphatidylinositol membrane anchor of acetylcholinesterase from the electric organ of the electric-fish, Torpedo californica.

Project description:The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) membrane anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.

| S-EPMC1137719 | biostudies-literature

Torpedo californica electric Organ Proteome

Project description:Bottom-up proteomics and mass spectometry were applied to determine the protein repertoire of the electric organ

2011-06-23 | PRD000388 | Pride

Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C.

Project description:The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.

| S-EPMC1152733 | biostudies-other

Soluble low-Km 5'-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage.

Project description:Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

| S-EPMC1132579 | biostudies-other

Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ.

Project description:Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (>63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes (C19orf29 [Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specialization using evolutionary diversity of organ function and cross-species mapping.

| S-EPMC3412124 | biostudies-literature

Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ.

Project description:The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1, N6-ethenoadenosine) 5',5'''-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (ApnA, where n=3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap(n-1). The Km values of the enzyme were 0.543+/-0.015, 0.478+/-0.043 and 0. 520+/-0.026 microM, and the Vmax values were 633+/-4, 592+/-18 and 576+/-45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5',5'''-P1,P3-triphosphate), Ap4A (adenosine 5',5"'-P1,P4-tetraphosphate) and Ap5A (adenosine 5',5'''-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86+/-0.11, 1.35+/-0.24 and 0.58+/-0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38+/-0.19 mM. The ATP analogues adenosine 5'-tetraphosphate and adenosine 5'-[gamma-thio]triphosphate are potent competitive inhibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29+/-0.03, 0.43+/-0.05 and 7.18+/-0.8 microM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7+/-3.1 microM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

| S-EPMC1218370 | biostudies-other

Phospholipid turnover in Torpedo marmorata electric organ during discharge in vivo.

Project description:One electric organ of anaesthetized Torpedo marmorata was stimulated through electrodes placed on the electric lobe of the brain. Nerves to the other electric organ were cut to provide an unstimulated control. Glucose 6-[32P]phosphate was injected into each organ 16h before electrical stimulation. After stimulation for 10 min at 5 Hz, the organs were removed homogenized and centrifuged on a density gradient for the preparation of subcellular fractions. Stimulation increased the incorporation of 32P into phosphatidate, phosphatidylinositol and phosphatidylcholine. The increased phosphatidate labelling, but not that of the other two lipids, was seen in fractions rich in synaptic vesicles. Stimulation had no effect on ATP labelling. The phosphatidate content of most fractions fell slightly after stimulation, but amounts of other phospholipids were not affected.

| S-EPMC1164010 | biostudies-other

Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction.

Project description:BackgroundDuring development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins.ResultsOf 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ?2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome.ConclusionsOur integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ.

| S-EPMC3156643 | biostudies-literature

Purification, characterization and cellular localization of 5'-nucleotidase from Torpedo electric organ.

Project description:5'-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5'-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.

| S-EPMC1148201 | biostudies-other

Association of the HNK-1 epitope with 5'-nucleotidase from Torpedo marmorata (electric ray) electric organ.

Project description:5'-Nucleotidase isolated from the electric organ of the electric ray (Torpedo marmorata) has a molecular mass of 62 kDa and, on two-dimensional electrophoresis, separates into up to 13 isoforms within a pI range of 5.9-6.7. The N-terminal sequence data show a 71% identity over 17 amino acids with that previously published for the rat liver enzyme. All forms of 5'-nucleotidase are recognized by the HNK-1 monoclonal antibody. HNK-1 immunoreactivity is found at the surface of the Schwann-cell processes covering the synaptic terminals and in this respect corresponds to that of 5'-nucleotidase in the same tissue. Since a number of glycoproteins involved in cell recognition and cell adhesion carry the HNK-1 epitope, 5'-nucleotidase may play a role in cell-cell or cell-extracellular matrix interaction in addition to its activity as an enzyme.

| S-EPMC1151468 | biostudies-other

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