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

ABSTRACT: 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.

SUBMITTER: Grondal EJ

PROVIDER: S-EPMC1148201 | biostudies-other | 1987 Aug

REPOSITORIES: biostudies-other

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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

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

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

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

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

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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.

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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.

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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.

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