Ecto-adenosine triphosphatase activity at the cholinergic nerve endings of the Torpedo electric organ

Synaptosomes isolated from the electric organ of Torpedo marmorata contain activity of an ATPase which is located at the extracellular face of the plasma membrane. Ecto-ATPase activity can be stimulated independently and to a similar extent by either Ca-2+ or Mg-2+. Apparent Km-values for ATP are 79 microM and 53 microM for Ca-2+ and Mg-2+ respectively. Apparent Km-values for Ca-2+ and Mg-2+ at 1 mM ATP are 0.71 mM and 0.61 mM respectively. The enzyme is also activated by Mn-2+ and GTP can replace ATP as a substrate. Presence of 5'- nucleotidase activity suggests that adenosine is the final hydrolysis product. Thus hydrolysis of nucleotides released during exocytosis of synaptic vesicle contents and purine salvage must be a major role of this ecto-enzyme. We furthermore suggest that the ecto-ATPase may provide the key to understanding the storage of the high energy compound ATP in cholinergic synaptic vesicles. On depolarization of the nerve terminal and exocytosis, ATP represents the signal for activating the ATPase whereby concentrations of Ca-2+ and Mg-2+ are already saturating. Following depolarization induced Ca-2+ influx, a possible function of the ATPase may be the outward transport of Ca-2+ from the nerve terminal.     

Carbohydrate patterns of the pure cholinergic synapse of Torpedo electric organ: a cytochemical and immunocytochemical electron microscopic approach.

Using post-embedding gold staining techniques, we investigated the ultrastructural distribution of terminal sugars and carbohydrate chains located at the pure cholinergic electric organ tissue of Torpedo marmorata. Neither alpha-N-acetylgalactosamine (GalNAc)-specific lectins (DBA, SBA, HPA) nor monoclonal antibodies (MAb) recognizing Tn (Gal-NAc alpha-O-Ser/Thr; MAb Cu-1) and sialyl-Tn epitopes (NeuAc alpha 2,6GalNAc alpha-O-Ser/Thr; MAb B72.3 and OSM-10) were capable of labeling any of the synaptic structures. The absence of gold labeling was likewise noted with UEA-I (L-fucose) and with PNA (T-antigen, Gal beta 1,3GalNAc alpha). After neuraminidase pre-treatment of ultra-thin sections, PNA labeling was rendered evident, indicating the presence of neuraminic acid-masked T-antigen. Certain synaptic vesicles were labeled for neuraminic acid (LFA) and for N-acetyllactosamine (DSA), whereas others were not labeled at all. Gold labeling with LFA, RCA-I (beta-galactose), and DSA in the membrane infoldings of the dorsal face of the electrocyte was visualized. As noted above, the PNA reaction was revealed only after pre-treatment with neuraminidase. Dorsal (non-synaptic) basal lamina were reactive with DSA, whereas the synaptic portion was likewise labeled with LFA and RCA-I. Finally, RCA-I labeling was noted in the Schwann cell nucleus. Comparisons between these results and those described at the neuromuscular junction were made.     

Characterization of phosphotyrosine containing proteins at the cholinergic synapse

Tyrosine phosphorylation has been associated with several aspects of the regulation of cholinergic synaptic function, including nicotinic acetylcholine receptor (AChR) desensitization as well as the synthesis and clustering of synaptic components. While some progress has been made in elucidating the molecular events initiating such signals, the downstream targets of these tyrosine kinase pathways have yet to be characterized. In this paper we have used molecular cloning techniques to identify proteins which are tyrosine phosphorylated at the cholinergic synapse. Phosphotyrosine containing proteins (PYCPs) were isolated from the electric organ of Torpedo californica by anti-phosphotyrosine immunoaffinity chromatography. Peptide sequencing and expression cloning then identified the isolated proteins. The proteins identified included heat shock protein 90, type III intermediate filament from Torpedo electric organ, alpha-fodrin, beta-tubulin, actin and rapsyn. These tyrosine phosphorylated proteins may play a role in the regulation of synaptic function by tyrosine kinases.     

Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

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

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.     

Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo

Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Characterization of desmin in the electric organ of Electrophorus electricus L

The intermediate filament protein of the electric organ from the Electrophorus electricus L. was purified in DEAE-cellulose column after extraction with a Triton X-100 buffer and urea solubilization. The desmin was analysed by SDS-PAGE against desmin purified from chicken gizzard. Characterization of desmin from the electric eel was carried out by peptide mapping and immunoblotting methods.     

A structural model of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures

Density measurements made on cholinergic synaptic vesicles from the electric organs of Torpedo marmorata at different osmotic pressures are consistent with the following structural model of the vesicle. The particle behaves like a sphere 80-100 nm in diameter bounded by a semi-permeable membrane. The bulk of its soluble constituents are in true solution at physiological osmolalities. The limiting membrane is approximately 4-5 nm thick, suggesting that it contains large areas of phospholipid bilayer exposed to its bathing medium. The limiting membrane takes up about 26% (v/v) of the particle, a further 34% (v/v) of which is osmotically active water and 31% (v/v) hydrated core material at 800 mosmol/1. The buoyant density of the membrane is 1.132 g . cm-3. The density of the hydrated core material is approximately 1.05 g . cm-3. The membrane is selectively permeable to small molecules when subjected to hypo-osmotic stress. It is proposed that this occurs by the formation of small transient pores in the lipid bilayer of the membrane, which are induced by stretching caused by the osmotic pressure change.     

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.     

G-proteins in Torpedo marmorata electric organ. Differential distribution in pre- and post-synaptic membranes and synaptic vesicles

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the alpha-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go alpha-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.     

Prefoldin recognition motifs in the nonhomologous proteins of the actin and tubulin families

Nascent actin and tubulin molecules undergo a series of complex interactions with chaperones and are thereby guided to their native conformation. These cytoskeletal proteins have the initial part of the pathway in common: both interact with prefoldin and with the cytosolic chaperonin containing tailless complex polypeptide 1. Little is understood with regard to how these chaperones and, in particular, prefoldin recognize the non-native forms of these target proteins. Using mutagenesis, we provide evidence that beta-actin and alpha-tubulin each have two prefoldin interaction sites. The most amino-terminally located site of both proteins shows striking sequence similarity, although these proteins are nonhomologous. Very similar motifs are present in beta- and gamma-tubulin and in the newly identified prefoldin target protein actin-related protein 1. Actin-related proteins 2 and 3 have related motifs, but these have altered charge properties. The latter two proteins do not bind prefoldin, although we identify them here as target proteins for the cytosolic chaperonin. Actin fragments containing the two prefoldin interaction regions compete efficiently with actin for prefoldin binding. In addition, they also compete with tubulins, suggesting that these target proteins contact similar prefoldin subunits.     

Further evidence that glycosaminoglycan specific to cholinergic synaptic vesicles recycles during electrical stimulation of the electric organ of Torpedo marmorata

Summary Semiquantitative immunohistochemical methods were used to demonstrate that at least some of the glycosaminoglycan contained within cholinergic synaptic vesicles is recycled during successive electrical stimulations of the electric organ of Torpedo marmorata.     

Periodic charge distribution in the intermediate filament proteins desmin and vimentin

A helical coiled-coil region of amino acid sequence surrounding the cysteine residue of desmin and vimentin shows a regular pattern of alternating positive and negative charges with periods close to $\text{28}\text{3}$ and $\text{28}\text{10}$ residues. This suggests relationships with the charge distributions of myosin rod and alpha-keratin. The common features may reflect a similar pattern of three-dimensional packing in vivo for each of these molecules.     

Development of the electromotor system in Torpedo marmorata: Cationic staining of the electric organ

Summary The electric organs of embryonic Torpedo marmorala have been reacted with three cationic stains to evaluate the appearance and distribution of anionic sites. Ruthenium red, alcian blue and lysozyme were used at different pHs and found to react in a time-related manner to anionic components within the interelectrocyte space. The basal lamina covering the ventral electrocyte surface possesses the greatest number of anionic sites whereas growth cone, presynaptic terminal and glial membranes displayed almost no staining. Since this lamina serves as the exclusive substrate for ingrowing neuntes during synaptogenesis, the results are consistent with the idea that charge distribution on the membrane surface may provide a necessary cue for neurite motility, extension and eventual synaptogenesis.     

Major non-histone proteins of bovine lymphocyte chromatin : Identification of tubulin and actin

Bovine lymphocyte chromatin contains a protein capable of binding colchicine suggesting that it is tubulin. Polyacrylamide gel electrophoresis of the chromatin proteins in the presence of sodium dodecyl sulfate (SDS) shows the presence of proteins co-migrating with tubulin and actin respectively. These two proteins are among the major protein constituents of the chromatin.     

Enkephalin degrading enzymes are present in the electric organ of Torpedo californica

Two proteolytic activities that degrade [Leu5]enkephalin were found in Torpedo californica electric organ. One is a soluble aminopeptidase that degrades enkephalin at the Tyr1-Gly2 peptide bond, and the second is an endopeptidase that degrades enkephalin at the Gly3-Phe4 peptide bond. The aminopeptidase is inhibited by low concentrations of puromycin and bestatin. More than 60% of the endopeptidase is associated with the particulate fraction and is almost completely inhibited by low concentrations of captopril (SQ 14225) or SQ 20881 (potent inhibitors of angiotensin converting enzyme). Thiorphan and phosphoramidon (potent enkephalinase inhibitors) are much less effective. The pattern of cleavage and inhibition of the particulate endopeptidase thus resembles that of angiotensin converting enzyme.