Observations on the innervated face of the electrocyte of the main organ of the electric eel (Electrophorus electricus L.)

Summary The innervated face of electrocytes in the main electric organ of Electrophorus electricus L. was examined by light microscopy, both conventional and with Nomarski contrast, and by transmission and scanning electron microscopy. Acetylcholinesterase cytochemistry was used in the demonstration of the greater density of synapses over the caudal papillae. The various techniques contributed to a better understanding of the distribution and form of papillae and synapses at the posterior face of the electrocyte. Caudal papillae are longer and thinner than those at the rostral face, but it was not possible to recognize a different type sometimes referred to in the literature as small papillae. The contact of nerve endings with the electrocyte seems to be made predominantly on the terminal half of caudal papillae, however a smaller number occur elsewhere on the posterior face. Synaptic terminals frequently appear as round profiles, but may be also elongated, with or without bulges, usually occupying a depression, and separated from the post-synaptic membrane by a space of 60–100 nm, where an expansion may be found.     

An ESR study of the postsynaptic membrane acetylcholinesterase of torpedo marmorata electric organ

Summary The effect of the cholinergic activator, phenyltrimethylammonium, on the ESR spectra of spinlabeled membrane bound acetylcholinesterase was studied; a reduction of maximal hyperfine splitting of the anisotropic ESR spectrum by 2 G was observed. The influence of phenyltrimethylammonium was prevented by the two cholinergic blocking agents d-tubocurarine and-cobratoxine. The present results indicate that the conformation change of the esteratic site of membrane acetylcholinesterase is triggered by the binding of phenyltrimethylammonium to the cholinoreceptor site.entjurcet al.: An ESR study of the postsynaptic membrane Acetylcholinesterase of torpedo marmorata electric organ.     

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.     

The A12 acetylcholinesterase and polypeptide composition of electric organ basal lamina of Electrophorus and some Torpedinae fishes

Basal lamina (BL) of Torpedo, Discopyge and Electrophorus electric organs was purified in order to establish polypeptide composition and association with acetylcholinesterase (AChE). Results indicate that BL presents a distinct peptide pattern and that the A12 form of AChE is directly attached to it. Comparison of the species studied demonstrated similarities both in polypeptide composition and AChE content of the purified BL. Extractions of BL with solutions of high ionic strength, guanidine-HCl and acetic acid indicated the differential solubilization of various domains of BL polypeptides.     

Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl₂ but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.     

Structural differences in the catalytic subunits of acetylcholinesterase forms from the electric organ of Torpedo marmorata

[3H]Diisopropylfluorophosphate was used to label covalently the catalytic subunits of the acetylcholinesterase forms extracted using different solubilization media. The incorporation of radiolabel was specific for true acetylcholinesterase, and SDS-polyacrylamide gel electrophoresis revealed that differences in molecular size existed between low salt-soluble (mol. wt. approximately 76 000), detergent-soluble (69 000) and high salt-soluble (72 000) acetylcholinesterase. These differences could not be attributed solely to an unusual migration behaviour but appeared to reflect differences in primary structure. While the basic unit of the low salt-soluble esterase was a monomer, the detergent-soluble esterase was linked by disulphide bridges to form dimers. The high salt-soluble form existed in large aggregates, whereby disulphide bridges form covalent links between the catalytic and non-catalytic elements. Pronase treatment showed that the differences were confined to the 'outer' structure of these molecules. The active site peptide exhibited homologies indicating that this part is conserved in the different classes of acetylcholinesterase. The results suggest that one can discriminate between at least three distinct esterase classes in the electric organ of Torpedo marmorata.     

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.     

The subcellular fractionation of the electric organ of Torpedo

Summary The acetylcholine-rich electric organ of Torpedo has been submitted to subcellular fractionation in an attempt to isolate nerve endings and synaptic vesicles derived from cholinergic neurones. Fractions containing small vesicles and granules as their only morphologically identifiable components also contained appreciable amounts of bound acetylcholine; however, it was not possible to demonstrate a specific enrichment of any one fraction with respect to bound acetylcholine as has been possible in brain. The tissue proved difficult to homogenize and few detached nerve endings (synaptosomes) were formed. A low-speed fraction rich in Na, K- activated adenosine triphosphatase contained numerous membrane fragments with tubular appendages derived from the non-innervated surface of electroplaques. Homogenization in media isotonic with elasmobranch plasma (e.g. 0.5 M sucrose + 0.33 M urea) was essential to preserve the structure of osmotically sensitive organelles (e.g. mitochondria).We wish to express our gratitude to Dr. R. D. Keynes who arranged the supply of Torpedos and to Mr G. H. C. Dowe and Miss L. Swales for skilled technical assistance. The electron microscopic facilities were provided by a grant from the Wellcome Trust and the work was supported by a grant no. NB-03928-02 (to V.P.W.) from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. During the period of this work Dr. Sheridan held a Postdoctoral Fellowship of the U.S. Public Health Service and Dr. Israël was an Exchange Scholar of the Medical Research Council.We are also most grateful to Professor Sir Bryan Matthews, C.B.E., Sc. D., F.R.S., for providing aquarium facilities in the Physiological Laboratory of Cambridge University.     

Hairy-root organ cultures for the production of human acetylcholinesterase

Background  

Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.     

Ontogeny of the electric organ discharge and the electric organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus (Hypopomidae, Gymnotiformes)

I recorded the electric organ discharges (EODs) of 331 immature Brachyhypopomus pinnicaudatus 6–88 mm long. Larvae produced head-positive pulses 1.3 ms long at 7 mm (6 days) and added a second, small head-negative phase at 12 mm. Both phases shortened duration and increased amplitude during growth. Relative to the whole EOD, the negative phase increased duration until 22 mm and amplitude until 37 mm. Fish above 37 mm produced a “symmetric” EOD like that of adult females. I stained cleared fish with Sudan black, or fluorescently labeled serial sections with anti-desmin (electric organ) or anti-myosin (muscle). From day 6 onward, a single electric organ was found at the ventral margin of the hypaxial muscle. Electrocytes were initially cylindrical, overlapping, and stalk-less, but later shortened along the rostrocaudal axis, separated into rows, and formed caudal stalks. This differentiation started in the posterior electric organ in 12-mm fish and was complete in the anterior region of fish with “symmetric” EODs. The lack of a distinct “larval” electric organ in this pulse-type species weakens the hypothesis that all gymnotiforms develop both a temporary (larval) and a permanent (adult) electric organ. Accepted: 1 March 1997