Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes.

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.

Are NPY and enkephalins costored in the same noradrenergic neurons and vesicles?

Separate studies show that NPY and enkephalins are widely distributed in peripheral noradrenergic neurons. In the present study, the subcellular costorage and release in response to intense sympathetic stimulation and reserpine at near therapeutic doses (0.05 mg/kg every other day) were examined. In young pig arteries and vas deferens, enkephalin and D beta H immunofluorescence show consistent but not total overlap. Also NPY is colocalized with D beta H in many fibers but with VIP (nonnoradrenergic) in others. Ultrastructural immunogold labeling indicates that individual terminals contain large dense cored vesicles (LDVs) which store either NPY or enkephalins, even though costorage of both peptides occurs. Some LDVs costore NPY and VIP, especially in the middle cerebral artery and in the lamina propria of vas deferens. Acute CNS ischemia depletes enkephalins and norepinephrine in all tissues analyzed without parallel loss of NPY. Reserpine depletes norepinephrine 70-85% but does not deplete NPY or enkephalins. The latter is in contrast to commonly used high doses known to produce nonspecific, detergent-like effects. In fact, low doses of reserpine induce a time-dependent new synthesis and processing of NPY precursor peptides in vas deferns. Contrasting effects of reserpine on NPY and enkephalin contents, new synthesis and apparent processing, and a differential response to acute CNS ischemia were found in every tissue studied. Activation of precursor neuropeptide processing occurred immediately upon intense sympathetic stimulation in most tissues. Dual localization of NPY in noradrenergic and nonnoradrenergic fibers and differences in subcellular LDV storage help explain why enkephalin correlates better than NPY with norepinephrine loss in response to acute CNS ischemia. Furthermore, the costorage of NPY and enkephalins in distinct subpopulations of noradrenergic fibers, which varies according to tissue, is likely to be under separate CNS control.

Depolarization regulates expression of a synaptic vesicle protein in rat superior cervical ganglia in vitro.

The role of membrane depolarization in the regulation of expression of a neuron specific protein was evaluated by culturing superior cervical ganglia from neonatal rats in defined medium and manipulating neuronal activity by depolarizing agents. P65 is an integral membrane protein of synaptic vesicles and can be used as a marker for general neuronal maturation. P65 antigen levels were quantified by indirect radioimmunoassay, using monoclonal antibodies. The expression of p65 in ganglion explants increased by 40-100% when the cultures were treated with the depolarizing agents, veratridine or high potassium. The veratridine effect could be blocked by simultaneous treatment with the sodium channel blocker, tetrodotoxin (TTX). The rise in p65 was not evident until 36 h after depolarizing treatment had begun and reached peak levels after 48 h, with no further increases observed with sustained treatment. After removal of the depolarizing treatment, p65 levels returned to control values after 24 h. P65 joins a growing number of molecules whose expression is regulated by membrane depolarization.

Characterization of synaptophysin and G proteins in synaptic vesicles and plasma membrane of Aplysia californica.

Two components of synaptic terminals that may be involved in transmitter release are synaptophysin (p38) and G proteins. In order to study release mechanisms in Aplysia californica we have prepared subcellular fractions from nervous tissue to characterize and localize these components. We identify Aplysia synaptophysin by Western blot analysis with monoclonal antibody SY38, find that it is enriched in synaptic vesicles, and, using immunocytochemistry, show that it is localized to neuropil. These characteristics indicate that Aplysia synaptophysin is closely related to mammalian synaptophysin; it appears to be much smaller, however, having a mass of 28 kDa instead of 38 kDa. We previously determined that G protein subunits in Aplysia are enriched in neuropil and synaptosomes. We now show that within the synaptic terminal the pertussis toxin-sensitive alpha-subunit as well as the beta-subunit are associated with plasma membrane using [32P]ADP-ribosylation and Western blotting with G protein-specific antibodies.

Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles.

Synaptophysin is a major integral membrane protein of synaptic vesicles. Its transmembrane topology deduced from the cDNA sequence predicts 4 transmembrane regions and a carboxy-terminal cytoplasmic tail containing a characteristic pentapeptide repeat structure. The monoclonal antibody (mAb), SY38, binds to a cytoplasmic domain of synaptophysin. By using fusion proteins corresponding to truncated forms of the cytoplasmic tail, its epitope was located to a flexible segment in the center of the repeat structure. Four other mAbs (c7.1, c7.2, c7.3, c7.4) share the same epitope, which thus emerges as the major immunogenic region of this membrane protein.

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals.

Nerve endings of the posterior pituitary are densely populated by dense-core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.

Purification and subunit composition of a cholinergic synaptic vesicle glycoprotein, phosphointermediate-forming ATPase.

A glycoprotein ATPase in cholinergic synaptic vesicles of Torpedo electric organ was solubilized with octa-ethylene glycol dodecyl ether detergent. Study of potential stabilizing factors identified crude brain phosphatidylserine, glycerol, dithiothreitol, and protease inhibitors as of value in maintaining activity. The ATPase was purified from the solubilized, stabilized material by glycerol density gradient band sedimentation velocity ultracentrifugation, and hydroxylapatite, wheat germ lectin affinity, and size exclusion chromatographies. The pure ATPase had a specific activity of about 37 mumol ATP hydrolyzed/min/mg protein. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified material typically exhibited three polypeptides of molecular masses 110, 104, and 98 kilodaltons (kDa) and a fourth diffuse polypeptide of 60 kDa. This composition suggests that the ATPase is a member of the P-type, or phosphointermediate-forming, family, but it was shown to be distinct from the ouabain-sensitive Na+,K+- and CA2+-stimulated Mg2+-ATPases. The purified vesicle enzyme was rapidly phosphorylated by [gamma-32P]ATP on about 14% of the subunits with molecular weights of 98,000-110,000. About 16% of the ATPase was phosphorylated in whole-vesicle ghosts in a manner consistent with formation of a phosphointermediate, thus confirming the P-type nature of this enzyme.

Characterization, by size, density, osmotic fragility, and immunoaffinity, of acetylcholine- and vasoactive intestinal polypeptide-containing storage particles from myenteric neurones of the guinea-pig.

When cytoplasmic extracts of guinea-pig myenteric neurones are submitted to centrifugal density gradient fractionation in a zonal rotor acetylcholine is bimodally distributed in the gradient, in a peak (I) rich in synaptic vesicles of the classic type and in a denser peak (II/VI) rich in densecored vesicles and vasoactive intestinal polypeptide (VIP). The putative stable synaptic vesicle markers synaptophysin (p38), vesicular proteoglycan, and Mg2+-activated ATPase were also bimodally distributed, with a peak coincident with peak I and another, broader peak embracing peak II/VI, and neighbouring peaks of other neuropeptides resolved from peak II/VI by the density gradient separation procedure used. To establish whether the stable markers, acetylcholine and VIP in peak II/VI were present in one particle or several, attempts were made to separate them by particle-exclusion chromatography and differential osmotic fragility. These were unsuccessful, leading us to conclude that the storage particles in peak II/VI contain both neurotransmitters and all three putative stable synaptic vesicle markers. It is suggested that such particles are the counterparts, in cholinergic neurones of the myenteric plexus, of the dense-cored, enkephalin- and noradrenaline-containing vesicles of certain adrenergic neurones and, like the latter, may exist in a precursor-product relationship with the classic synaptic vesicles containing the small-molecular-mass transmitters and found in the same nerve terminals.

Modification of vesicular dopamine and norepinephrine by monoamine oxidase inhibitors.

The possible effects of inhibitors of the two forms of monoamine oxidase (types A and B) on dopamine (DA) and norepinephrine (NE) accumulation and metabolism in the cytoplasmic and microsomal (vesicular) fractions of the rat brain have been examined. It was found that, while L-DOPA treatment raised only cytoplasmic DA without affecting vesicular DA and NE, clorgyline and pargyline treatments caused significant increases in DA and NE concentrations in both cytoplasmic and vesicular fractions. The DA increase in the synaptic vesicles (200-600%) was much more pronounced than that (150%) in the cytoplasm. In contrast, deprenyl treatment increased vesicular DA only slightly without any effect on either vesicular or cytoplasmic NE. L-DOPA administration to rats pretreated with clorgyline and pargyline, but not with deprenyl, further increased cytoplasmic and vesicular DA and NE concentrations. However, excessive increases in vesicular DA lowered vesicular NE. Reserpine drastically reduced vesicular and cytoplasmic DA and NE, and L-DOPA administration to the reserpine-treated rats caused a DA increase only in the cytoplasmic fraction without affecting vesicular DA or NE. The effect of reserpine was abolished by pargyline treatment, which suggests that pargyline may interact with the reserpine-sensitive vesicular uptake. There was a significant correlation between vesicular DA and NE increase.

Most synaptic vesicles isolated from rat brain carry three membrane proteins, SV2, synaptophysin, and p65.

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.

A synaptic vesicle membrane protein is conserved from mammals to Drosophila.

The structure of synaptobrevin, an intrinsic membrane protein of small synaptic vesicles from mammalian brain, was studied by purification and molecular cloning. Its message in bovine brain encodes a 116 amino acid protein whose sequence reveals it to be the mammalian homolog of Torpedo VAMP-1. Antibody probing demonstrates that the protein is also present in Drosophila, and its Drosophila homolog was cloned. Alignment of the sequences of synaptobrevin/VAMP-1 from the three species shows it to contain four domains, including a highly conserved central region of 63 amino acids that contains 75% invariant residues. The finding that a membrane protein from vertebrate synaptic vesicles is conserved in Drosophila points toward a central role of this protein in neurotransmission and should allow a genetic approach to neurotransmitter release.

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.

A silver-reducing component in rat striated muscle. II. Isolated sarcoplasmic reticulum vesicles.

In previous work on rat striated muscle cells a silver-reducing component was found selectively localized at the terminal cistern/transverse tubule system (Tandler and Pellegrino de Iraldi 1989). To further investigate that problem we performed the Hg-Ag argentaffin reaction on a sarcoplasmic reticulum fraction from rat skeletal muscle. Circular profiles corresponding to vesicular structures were found outlined by silver grains. The number of silver "stained" vesicles were less than the total number vesicles stained by conventional procedures. The correlation between argentaffinities in the intact muscle fiber and their subcellular organelles indicated that the Hg-Ag reactive vesicles must be those derived from the terminal cisternae of the sarcoplasmic reticulum. The silver-reducing constituent aggregates in the presence of 1 mM CaCl2 or 0.5 M K cacodylate. The state of aggregation induced by Ca2+ was not affected by incubation with 0.5% Triton X-100 or by 2 mM EDTA, thus suggesting a localization at or near the membrane of the terminal cistern vesicle facing the junctional gap. In Laemmli SDS-acrylamide gels the Hg-Ag reaction stained all proteins in a manner similar to Coomasie blue. It is suggested that the selective histochemical staining is the result of differential reactivities due to steric requirements of the chemical reaction.

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1.

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

Light and electron microscopical studies of the GABA innervation of the dorsal column nuclei and the lateral cervical nucleus in the primate species Macaca fascicularis and Papio anubis.

In 4 monkeys of the species Macaca fascicularis (2 animals) and Papio anubis (2 animals) the three dorsal column nuclei and the lateral cervical nucleus have been investigated immunocytochemically with antiserum against gamma-aminobutyric acid (GABA). Light microscopic studies demonstrated the presence of GABA-positive cells in the gracile nucleus, the internal cuneate nucleus and the lateral cervical nucleus but not in the external cuneate nucleus. Although labeled cells seemed fairly evenly spread in the nuclei there was an increased amount between the clusters of the internal cuneate nucleus and in the border zone between the gracile and the cuneate nucleus. Electron microscopical investigation showed GABA labeling in fairly small neurons with relatively large cell nuclei and low somatic bouton covering. GABA-positive terminals with rounded synaptic vesicles were present in all the investigated nuclei also the external cuneate one. No apparent difference in number of such boutons in the different nuclei or parts of the nuclei was found. GABA-positive boutons mostly synapsed with dendrites but in the dorsal column nuclei also with other larger boutons. Axosomatic contacts between labeled terminals and neuronal perikarya were more common in the lateral cervical nucleus than in the dorsal column nuclei. The results from the different nuclei in the monkey were compared with the results of similar investigations in the cat. It is concluded that there are important species differences especially on the light microscopical level in the lateral cervical nucleus. Thus the GABA labeled cells is rather evenly spread over the nucleus in the monkey whereas in the cat they are concentrated to the ventromedial region.

Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38.

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.

In adrenal medulla synaptophysin (protein p38) is present in chromaffin granules and in a special vesicle population.

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells.

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.