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.     

Immunohistochemical localization of a synaptic-vesicle antigen in a cholinergic neuron under conditions of stimulation and rest

Summary An antiserum against a specific component (a glycosamino glycan) of the cholinergic synaptic-vesicle of Torpedo marmorata has been used to investigate the localization of the component in the cell body, its movement within the electromotor axon and its fate within the nerve terminal upon electrical stimulation. After immunofluorescent staining, spots are observed throughout the cytoplasm of the lobe perikarya, although they are concentrated in the region of the axon hillock. Ligation of the electromotor nerves leading from the lobe to electric organ produces a proximal build-up of material which stains readily with the antivesicle antiserum, indicating that the vesicle antigen is transported from the cell body to the nerve terminal. A marked increase in indirect immunofluorescent staining of the electric organ is observed in the nerve ending upon electrical stimulation. We interpret this result as fusion of the vesicles with the presynaptic plasma membrane and exteriorization of the vesicle antigen to the extracellular space, thereby facilitating its staining. After recovery of the system the fluorescence declines, a result that is consistent with the reinternalization of the vesicle antigen into the core of reformed vesicles. The results support a mechanism whereby vesicles recycle within the nerve terminal and transmitter is released by exocytosis.     

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.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)     

Ganglioside composition of subcellular fractions,including pre- and postsynaptic membranes,fromTorpedo electric organ

Gangliosides were isolated from four subcellular fractions of the electric organ ofTorpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes. This exploited a principal advantage offered by this tissue: facile separation of pre-and postyynaptic elements. Total ganglioside concentration in presynaptic membranes was approximately twice that of synaptosomes and 15 times that of postsynaptic membranes (47.7, 24.4, and 3.21 g of lipid sialic acid per mg protein, respectively). Synaptic vesicle membranes had the highest overall concentration (78.9) relative to protein, but a concentration approximately comparable to that of presynaptic membranes when expressed relative to phospholipid. The thin-layer patterns of these two fractions were similar, both in terms of total pattern and the specific pattern of gangliotetraose structures as revealed by overlay with cholera toxin B subunit; these were notable for the paucity of monosialo structures and the virtual absence of GM1. Postsynaptic membranes, on the other hand, had a significantly higher content of monosialogangliosides including the presence of GM1. The synaptosomal pattern resembled that of the presynaptic membranes and synaptic vesicles. Thus, a clear difference in ganglioside pattern could be discerned between the pre- and postsynaptic elements of the electric organ.Abbreviations SVs synaptic vesicles - TLC thin-layer chromatography - cholera B-HRP B subunit of cholera toxin linked to horseradish peroxidase     

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components

Summary Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N -ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.     

Immunohistochemical localization of cholinergic nerve terminals

Summary Most of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.     

Structural changes at pure cholinergic synaptosomes during the transmitter release induced by A-23187 inTorpedo marmorata

Summary Pure cholinergic synaptosomes isolated from the electric organ ofTorpedo marmorata were stimulated by calcium ionophore A-23187. The effect of time course of stimulation on the changes in intramembrane particles (IMPs) on presynaptic membranes was studied by quickfreezing and aldehyde-fixation freeze-fracture. We showed that the decrease of small-particle density at the P-face and the increase of large-particle density at the E-face was maximum after 30 sec of A-23187 stimulation. Later, the density of synaptic vesicles decreased. We suggest that the redistribution of IMPs on the presynaptic membrane and acetylcholine (ACh) release from pure cholinergic synaptosomes have a similar time course when triggered by A-23187     

Isolation of Cholinergic Synaptic Vesicles from the Myenteric Plexus of Guinea-Pig Small Intestine

The acetylcholine-rich myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine has been subjected to subcellular fractionation using modifications of both classical methods and that originally devised for bulk isolation of cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo marmorata by means of density gradient centrifugation in a zonal rotor. The latter method gave a vesicle fraction with the highest acetylcholine content so far recorded for a mammalian particulate fraction, 30.9 × S.E.M. 1.8 (5) nmol of acetylcholine × mg of protein^?1. Electron-microscopical examination showed that it consisted of a homogeneous preparation of vesicles of mean spherical diameter 61 ×sd 4 (108) nm, with little or no contamination with other lipoprotein membrane structures, mixed how-ever with considerable amounts of actomyosin fibrils, presumably derived from the longitudinal muscle. Slab-gel electrophoresis in sodium dodecyl sulphate showed that, in addition to prominent peaks attributable to actin and myosin, there was a relatively simple pattern of (presumably) vesicle protein among which all the proteins thought to be characteristic of Torpedo synaptic vesicles were present. Dowe G. H. C. et al. Isolation of cholinergic synaptic vesicles from the myenteric plexus of guinea-pig small intestine. J. Neurochem. 35, 993–1003 (1980).     

Effects of ouabain and electrical stimulation on the fine structure of nerve endings in the electric organ of Torpedo marmorata

Summary The cycle of synaptic vesicles was studied in isolated nerve terminals and in the electric tissue of Torpedo marmorata. The synaptosomes, as used in this investigation, were a pure cholinergic subcellular fraction that captured dextran particles as an extracellular marker. This endocytotic phenomenon was enhanced by potassium depolarization. Field electrical stimulation (1 Hz and 10 Hz) of the electric organ induced the appearance of membrane foldings into presynaptic terminals. Morphometric studies showed that the number of synaptic vesicles did not decline until after at least 30 min. On the other hand, at 10 Hz these changes were accompanied by an increase in length of the membrane of the terminal. At 15 min of recovery after prolonged stimulation, there was a great increase in density of synaptic vesicles with a large number of vesicles of small diameter. This increase was accompanied by a decrease of membrane length, suggesting that reformation of vesicles is related to retrieval of membrane. Pharmacological stimulation with ouabain produced changes similar to those of long-term electrical stimulation. These changes in membrane were accompanied by a decrease of the population of synaptic vesicles and a wide variation in their diameters. It is concluded that structural changes reported here could not be correlated with kinetics of the transmitter release.We are grateful to Dr. E. Cañadas, Prof. Dr. D. Ribas and Dr. J. Tomás for valuable help and encouragement. We are indebted to Dr. P. Arté and to the staff of the Acuario de Barcelona del Instituto de Investigaciones Pesqueras for providing specimens of Torpedo marmorata. This investigation was supported by a grant Formación Personal Investigador del Ministerio de Universidades e Investigación     

Identification of a Proteoglycan Antigen Characteristic of Cholinergic Synaptic Vesicles

An antiserum to cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata was purified by adsorption with fractions containing unwanted antigens. The adsorbed antiserum responds to the proteoglycan core material of the cholinergic synaptic vesicles. The major antigen migrates in an anomalous fashion on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), forming a broad band with an apparent molecular weight of approximately 120,000 - 300,000. The distribution of this antigen after sucrose density gradient centrifugation of synaptic vesicles is the same as that of vesicular ATP. The antigen comigrates with a substance that can be stained with Alcian-Blue after SDS-PAGE of highly purified synaptic vesicles. This substance is related to the low-molecular-weight, Alcian-Blue-positive glycosaminoglycan vesiculin, which is formed from the high-molecular-weight proteoglycan by prolonged dialysis against water or by protease treatment. No antibodies were detected against vesiculin itself, indicating that the antigenic determinants are restricted to the proteoglycan.     

DIFFERENT RECOVERY RATES OF THE ELECTROPHYSIOLOGICAL, BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS IN THE CHOLINERGIC SYNAPSES OF THE TORPEDO ELECTRIC ORGAN AFTER STIMULATION

—During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re-established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re-established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.     

Recycled Synaptic Vesicles Contain Vesicle but Not Plasma Membrane Marker, Newly Synthesized Acetylcholine, and a Sample of Extracellular Medium

To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low-frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate-dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extracellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low-frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata were found to contain a proteoglycan in their core. The glycosaminoglycan part co-migrates upon thin layer electrophoresis with heparan sulphate and shows a chemical composition characteristic for this carbohydrate. [35S]Sulphate injected into the electric lobes of Torpedo, which contain the perikarya of the electromotor neurons innervating the electric organs, appeared 48 h later in covalently bound form in the synaptic vesicle fraction. The radiolabel had been incorporated into the vesicular heparan sulphate. Upon SDS-polyacrylamide gel electrophoresis fluorography of labelled vesicles a major and a minor band are formed both migrating above a protein standard of mol. wt. 200 000. Similarly, a major peak in the void volume and a minor peak in the included volume are seen upon gel filtration in Ultrogel AcA 34 in the presence of SDS. We interpret the minor fraction as being formed by the loss of glycosaminoglycan from the major fraction. The proteoglycan is located inside the vesicle since antibodies directed against it form immunoprecipitates only with vesicles lysed by detergent treatment. The experiments show that it is possible to label a synaptic organelle specifically by axonal transport.     

The Synaptic Vesicle-Associated G Protein o-rab3 Is Expressed in Subpopulations of Neurons

Abstract: The distribution of o-rab3—a synaptic vesicle-associated low-molecular-weight GTP-binding protein—was studied in various neural tissues of the electric ray Torpedo marmorata. o-rab3 was shown to be associated selectively with isolated cholinergic synaptic vesicles derived from the electric organ. Gel filtration of cholinergic synaptic vesicles using Sephacryl S-1000 column chromatography demonstrated a copurification of o-rab3 with the synaptic vesicle content marker ATP and with SV2—a synaptic vesicle transmembrane glycoprotein. Indirect immunofluorescence using antibodies against o-rab3 and SV2 and a double labeling protocol revealed an identical distribution of both antigens in the cholinergic nerve terminals within the electric organ and at neuromuscular junctions. An immunoelectron microscopic analysis demonstrated the presence of o-rab3 at the surface of the synaptic vesicle membrane. In the CNS immunofluorescence of o-rab3 and SV2 overlap only in small and distinct areas. Whereas SV2 has an overall distribution in nerve terminals of the entire CNS, o-rab3 is restricted to a subpopulation of nerve terminals in the dorsolateral neuropile of the rhombencephalon and in the dorsal horn of the spinal cord. Our results demonstrate that the synaptic vesicle-associated G protein o-rab3 is specifically expressed only in subpopulations of neurons in the Torpedo CNS.     

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-(14)C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32+/-(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.