Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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.

ABSTRACT: 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.

SUBMITTER: Vogel M

PROVIDER: S-EPMC1132579 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

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

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

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

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

Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.

Project description:Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.

| S-EPMC1131318 | biostudies-other

Isolation of pure cholinergic nerve endings from the electric organ of Torpedo marmorata.

Project description:A rapid method for the preparation of highly purified cholinergic nerve endings from the electric organ of Torpedo is described. The endings retain their cytoplasmic components, as shown by biochemical and morphological observations. The homogeneity of these synaptosomes make them a useful tool for further studies.

| S-EPMC1164206 | biostudies-other

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

cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.

Project description:Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

| S-EPMC1149627 | biostudies-other

Subcellular localization and ionic properties of acetyl-coenzyme A synthetase in the electric organ of Torpedo marmorata.

Project description:Acetyl-CoA synthetase activity was shown to be present in pure cholinergic synaptosomes from electric organ of Torpedo marmorata. After osmotic disruption of synaptosomes a substantial part of the activity was recovered in the soluble fraction. The effects of varying pH and increasing K+ concentrations on the synaptosomal enzyme activity were shown to differ from those observed with the mitochondrial enzyme. Whereas this latter enzyme showed optimal activity above pH 8.5, and a maximal activation in the presence of 120 mM-K+, the synaptosomal enzyme exhibited an optimal activity at pH 7.9 and a moderate K+ stimulatory effect with an optimal concentration of 30 mM.

| S-EPMC1161053 | biostudies-other

Biosynthesis of acetyl-coenzyme A in the electric organ of Torpedo marmorata in relation to acetylcholine metabolism.

Project description:Formation of acetyl-CoA through acetyl-CoA synthetase (forward reaction) and through choline acyltransferase (backward reaction) was investigated in tissue extract from the electric organ of Torpedo marmorata. When the tissue extract was submitted to gel filtration on Sephadex G-25, the formation of acetyl-CoA by acetyl-CoA synthetase appeared fully dependent on ATP and CoA and partially dependent on acetate (an endogenous supply of acetate is discussed). Choline acetyltransferase was a potent source of acetyl-CoA, only requiring acetylcholine and CoA, and was much more efficient than acetyl-CoA synthetase for concentrations of acetylcholine likely to be present in nerve endings.

| S-EPMC1165028 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data