Synaptic vesicles in electromotoneurones. I. Axonal transport, site of transmitter uptake and processing of a core proteoglycan during maturation.

We were able by using an in vivo pulse-label technique to trace part of the life cycle of a secretory organelle, the acetylcholine-storing synaptic vesicle from electromotoneurones of Torpedo marmorata. This technique uses [35S]sulphate incorporation into the cell bodies of the electromotoneurones which results in radioactive labelling of a synaptic vesicle heparansulphate proteoglycan--a major core component. Vesicles are anterogradely transported in the axons at a fast rate as 'empty' organelles (VP0 population). In the nerve terminal, maturation of the granule to a population (VP1) fully charged with acetylcholine and ATP occurs. Finally after a longer time interval a change to a third population (VP2) is observed. This population is reduced in diameter as compared to VP0 and VP1 suggesting, in agreement with earlier reports, that it has undergone exo-endocytosis. The changes from VP0 to VP1 and VP2 are accompanied by a degradation of the core proteoglycan as measured by gel filtration of the 35S-labelled compound. The results show that vesicles are axonally transported as preformed organelles, exist in the neurone at least in three different populations and that the nerve terminal is the major site of transmitter uptake.     

Changes in nuclear DNA and RNA during epidermal keratinization

Summary The morphology of the oval nucleus of neonatal Torpedo marmorata is described at the light and electron microscopic level of examination. The nucleus is unique relative to other central electromotor centers of electric fish so far described being bilaterally symmetrical, composed of two nerve cell types, and possessing no gap junctions between neurons and their processes. This particular structural plan presents difficulties in accounting for presumed synchronous discharge since it has been strongly argued that electrotonic coupling by means of gap junctions is the primary process by which synchronization is accomplished. Close membrane apposition and dendritic bundling, common features within the nucleus, are discussed as possible alternative structural correlates.     

Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus

We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.     

Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Purification and Characterization of a Peptidyl Dipeptidase Resembling Angiotensin Converting Enzyme from the Electric Organ of Torpedo marmorata

The electric organ of Torpedo marmorata contains a membrane-bound, captopril-sensitive metallopeptidase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chromatography using the selective inhibitor lisinopril immobilised to Sepharose via a 28-A spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C-terminus of a variety of peptide substrates, including angiotensin I, bradykinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)-His-Leu. The hydrolysis of BzGly-His-Leu was activated by Cl-. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuropeptide substance P. Hydrolysis of substance P occurred primarily at the Phe8-Gly9 bond with release of the C-terminal tripeptide, Gly-Leu-MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr = 190,000) than the pig kidney enzyme (Mr = 180,000) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.     

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.     

Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle.

Polypeptide chains composing acetylcholine receptors from the electric organs of Torpedo californica and Electrophorus electricus were purified and labeled with 125I. Immunochemical studies with these labeled chains showed that receptor from Electrophorus is composed of three chains corresponding to the alpha, beta, and gamma chains of receptor from Torpedo but lacks a chain corresponding to the delta chain of Torpedo. Experiments suggest that receptor from mammalian muscle contains four groups of antigenic determinants corresponding to all four of the Torpedo chains. Binding of 125I-labeled chains was measured by quantitative immune precipitation and electrophoresis. Antisera to the following immunogens were used: denatured alpha, beta, gamma, and delta chains of Torpedo receptor, native receptor from Torpedo and Electrophorus electric organs and from rat and fetal calf muscle, and human muscle receptor (from autoantisera of patients with myasthenia gravis). The four chains of Torpedo receptor were immunologically distinct from one another and from higher molecular weight chains found in electric organ membranes. Antibodies to these chains reacted very efficiently with native Torpedo receptor, but the reverse was not true. Antibodies to native receptor from Torpedo and Electrophorus reacted slightly with each of the chains of the corresponding receptor. However, cross-reaction between chains and antibodies to any native receptor was most obviuos with the alpha chain of Torpedo or the corresponding alpha' chain of Electrophorus. Antiserum to alpha chains exhibited higher titer aginst receptor from denervated rat muscle. Antibodies from myasthenia gravis patients did not cross-react detectably with 125I-labeled chains from electric organ receptors. Most interspecies cross-reaction occurred at conformationally dependent determinants whose subunit localization could not be determined by reaction with the denatured chains.     

Role of apoptosis and Fas/FasL system in the oogenesis of the spotted ray Torpedo marmorata

In the present paper we investigated the role played by apoptosis during oogenesis in the cartilaginous fish Torpedo marmorata. TEM, TUNEL and immunohistochemical techniques were employed to specifically reveal morphological and biochemical hallmarks of apoptosis in specimens from birth to sexual maturity. Data obtained demonstrate that apoptosis occurs in prefollicular oocyte selection, in maintaining the homeostasis of granulosa in healthy growing oocyte and in resorbing atretic follicles. In this respect, the involvement of apoptosis in Torpedo marmorata oogenesis closely parallels that found in mammals, thus confirming that strategies of germ cell selection among vertebrates have been evolutionarily preserved.     

Immunological comparison of acetylcholine receptors and their subunits from species of electric ray.

Rabbit antibodies were produced against purified acetylcholine receptor and each of the four acetylcholine receptor subunits from Torpedo californica. Using the technique of double diffusion in agar, cross reactivities were observed between these antibodies and purified acetylcholine receptor and receptor subunits from Torpedo marmorata, Torpedo nobiliana, and Narcine brasiliensis, as well as from Torpedo californica. The specificity of each of the four anti-subunit antibodies and the conservation of subunit antigenic determinants in the four electric rays studied are demonstrated.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Substitution of Torpedo acetylcholine receptor alpha 1-subunit residues with snake alpha 1- and rat nerve alpha 3-subunit residues in recombinant fusion proteins: effect on alpha-bungarotoxin binding.

A fusion protein consisting of the TrpE protein and residues 166-211 of the Torpedo acetylcholine receptor alpha 1 subunit was produced in Escherichia coli using a pATH10 expression vector. Residues in the Torpedo sequence were changed by means of oligonucleotide-directed mutagenesis to residues present in snake alpha 1 subunit and rat nerve alpha 3 subunit which do not bind alpha-bungarotoxin. The fusion protein of the Torpedo sequence bound 125I-alpha-bungarotoxin with high affinity (IC50 = 2.5 x 10(-8) M from competition with unlabeled toxin, KD = 2.3 x 10(-8) M from equilibrium saturation binding data). Mutation of three Torpedo residues to snake residues, W184F, K185W, and W187S, had no effect on binding. Conversion of two additional Torpedo residues to snake, T191S and P194L, reduced alpha-bungarotoxin binding to undetectable levels. The P194L mutation alone abolished toxin binding. Mutation of three Torpedo alpha 1 residues to neuronal alpha 3-subunit residues, W187E, Y189K, and T191N, also abolished detectable alpha-bungarotoxin binding. Conversion of Try-189 to Asn which is present in the snake sequence (Y189N) abolished toxin binding. It is concluded that in the sequence of the alpha subunit of Torpedo encompassing Cys-192 and Cys-193, Try-189 and Pro-194 are important determinants of alpha-bungarotoxin binding. Tyr-189 may interact directly with cationic groups or participate in aromatic-aromatic interactions while Pro-194 may be necessary to maintain a conformation conductive to neurotoxin binding.     

Na(+)-Ca2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata.

Synaptic plasma membranes obtained by hypo-osmotic treatment of purified Torpedo ocellata synaptosomes, contain an electrogenic Na(+)-Ca2+ exchange system. The dependence of the initial reaction rate on [Ca2+] reveals a single binding site for Ca2+ with an average apparent Km of 13.66 (S.D. = 12.07) microM [Ca2+] and maximal reaction velocity of Vmax = 11.33 (S.D. = 5.93) nmol/mg protein per s. The dependence of the initial rate of the Na+ gradient dependent Ca2+ influx on the internal [Na+] exhibits a sigmoidal curve which reaches half-maximal reaction rate at 170.8 (S.D. = 19.9) mM [Na+]. Addition of ATP gamma S does not change the K0.5 to Na+. The average Hill coefficient is 3.09 (S.D. = 0.86) indicating that 3-4 Na+ ions are exchanged for each Ca2+. Na+ gradient dependent Ca2+ uptake in Torpedo SPMs takes place also in the absence of K+ suggesting that K+ co-transport is not obligatory. The temperature dependence of the initial and steady-state rates of Na+ gradient dependent Ca2+ influx reveal that maximal reaction velocities of the Torpedo exchanger are attained between 15 and 20 degrees C. The energy of activation between 0 and 20 degrees C is 20,826 cal/mol. In comparison, rat brain synaptic plasma membrane Na(+)-Ca2+ exchanger reaches maximal reaction rates between 30 and 40 degrees C. Reconstitution of Torpedo or rat brain Na(+)-Ca2+ exchangers into a membrane composed of either Torpedo or brain phospholipids, does not alter the temperature dependence of the native Torpedo or rat brain Na(+)-Ca2+ exchangers; inspite of considerable differences in the composition of the fatty acyl chains that are esterified to brain and Torpedo phospholipid head groups and differences in membrane fluidity that were detected. An ATP-dependent Ca2+ pump, which is insensitive to FCCP, is also present in the same synaptic membrane.     

Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.     

α-Bungarotoxin Binding in House Fly Heads and Torpedo Electroplax

Abstract: House fly heads contain a site that binds α-bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds α-bungarotoxin (labeled with [³H]pyridoxamine phosphate) reversibly with a K _d of 6 nM. The effects of 48 drugs have been compared on the α-bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly site is similar to that previously reported for neuronal α-bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl-β-methylcholine, but lower affinities for nicotine, atropine, and dihydro-β-erythroidine.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

Nicotinic acetylcholine receptor: a structural model for alpha-subunit peptide 188-201, the putative binding site for cholinergic agents

A peptide corresponding to amino acid sequence 188-201 of the alpha-subunit of Torpedo AChR binds alpha-Bgtx. The S-S bridge between Cys 192 and 193 is essential for the binding as Tyr in position 189. The same sequence 188-201 corresponding to human AChR, which instead of Tyr has a Thr in position 189, binds alpha-Bgtx with a much lower efficiency. Monoclonal antibodies raised against Torpedo peptide 188-201 recognize Torpedo AChR and antibodies against Torpedo AChR recognize peptide 188-201 indicating that the synthetic peptide and the corresponding sequence in the native molecule share some immunological epitopes. With computer graphics and energy refinement a molecular model of this peptide has been elaborated.     

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.