The high-molecular-weight proteins of bovine brain plain synaptic vesicles

High molecular mass polypeptides (M _r >100,000) of plain synaptic vesicles from bovine cerebral cortex were separated using porous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Four major bands, ofM _r 262,000, 249,000, 216,000, and 173,000, were resolved. Investigations into the membrane association of theM _r 216,000 and 173,000 proteins by means of solubilization experiments and Sepharose 4B chromatography indicate that the former is a peripheral protein and the latter is more firmly attached, possibly an integral protein. Finally, theM _r 216,000 protein was shown to be highly enriched in synaptic vesicles compared to other brain subfractions. It thus appears to be specifically associated with synaptic vesicles and therefore may have an important role specific to synaptic vesicle function or structure.     

Characterization of DIDS-sensitive ATP accumulation in brain synaptic vesicles

ATP is an excitatory neurotransmitter in the central and peripheral nervous system. We investigated ATP accumulation in highly purified brain synaptic vesicles (SVs). Based on the amount of ATP accumulated in SVs under the conditions used, ATP is not transported against a concentration gradient but rather appears to have a Delta mu H(+)-independent mechanism. ATP transport was inhibited by DIDS and NEM, but was not affected by Mg(2+) or by pre-incubation with nucleotides. These results suggest a unique transport mechanism that does not involve exchange with other nucleotides or protons, unlike other known neurotransmitter transport systems.     

Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes

The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as "lipid rafts," has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as 'lipid rafts.' By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-beta-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.     

The uptake of horseradish peroxidase by cortical synapses in rat brain

Summary Horseradish peroxidase (HRP) was introduced directly into the cerebral cortex of adult rats, which were allowed to survive for 60 min before perfusion fixation. After the tissue had been incubated to demonstrate HRP at the LM and EM levels, blocks of cortical tissue were taken at varying distances from the injection site. These eight blocks of tissue constituted a time sequence for HRP diffusion.Qualitative examination of the presynaptic terminals showed that the most commonly encountered profiles are the plain synaptic vesicles, many of which accumulate tracer. In some terminals labelled vesicles are lined-up in tubular fashion. Other profiles commonly labelled are coated vesicles, tubular and vacuolar cisternae, and plain and coated pinocytotic vesicles.Quantitative analyses based on the number of terminals containing labelled profiles demonstrate an early rise in the rate of labelling of both plain synaptic vesicles and coated vesicles, after which synaptic vesicle labelling rises slowly towards a plateau. By contrast, there is a late parallel increase in the rate of labelling of coated vesicles and cisternae. A more detailed analysis, based on the actual numbers of labelled and total profiles within each presynaptic terminal, highlight early and late periods of rapid labelling for plain synaptic vesicles, coated vesicles and cisternae. A further aspect of HRP incorporation studied, concerns its uptake into four delineated regions of the presynaptic terminal.Our data indicate that membrane uptake into the presynaptic terminal is accomplished mainly via coated vesicles, although plain synaptic vesicles may also be involved. Coated vesicles, in turn, appear to give rise directly to plain synaptic vesicles, with some coalescing to produce vacuolar cisternae. The latter are involved in a two-way interchange of membrane with tubular cisternae, plain synaptic vesicles and coated vesicles. An additional source of plain synaptic vesicles are the tubular cisternae. Exocytosis of plain synaptic vesicles constitutes the mechanism by which transmitter is released from the presynaptic terminal.Supported by the Nuffield Foundation. We are grateful to Mr. M. Austin for help with the photography     

ZIO staining in synaptic vesicles of the rat pineal nerves after inhibition of serotonin and noradrenaline synthesizing enzymes

Summary Two compartments have been defined in monoaminergic synaptic vesicles: the core or central compartment, storage site for monoamines, and the matrix or outer compartment, of unknown function. The outer compartment reacts with the mixture of zinc iodide-osmium tetroxide (ZIO). This reaction is temperature and time dependent and may be abolished by -SH reagents.The effect of drugs inhibiting the synthesis of serotonin and noradrenaline (stored in the core) on the ZIO reaction in the matrix was studied in synaptic vesicles of rat pineal nerves. The inhibitors of monoamine synthesis abolish or decrease the ZIO reaction directly or in combination with the administration of tyramine. This effect is temperature dependent suggesting that the drugs act on different components of the matrix that react with ZIO at different temperatures. A comparison of the present results with those obtained with -SH reagents seems to indicate that the drugs assayed act, at least in part, by changing the accessibility of-SH groups in vesicle proteins. (An abstract of this paper was presented at the 7th International Congress of Pharmacology, Paris, 1978.)Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, CONICET, and the Secretaría de Ciencia y Técnica, SECYT, ArgentinaWe are grateful to Margarita López de Cáceres for technical assistance, to Adriana Contreras for the electron micrographs and to María Aued de Rau for the illustrations. Rita Cardoni is a fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

On the distribution of axonal terminals containing spheroidal and flattened synaptic vesicles in the hippocampus and dentate gyrus of the rat and cat

Summary A quantitative analysis has been made of the distribution of presynaptic profiles containing round (or spheroidal) and flattened (or ellipsoidal) synaptic vesicles in the apical and basal dendritic zones and in the layer of pyramidal cell somata of fields CA₁ and CA₃ of the hippocampus, and in the molecular and granular layers of the dentate gyrus of the rat and cat.In the apical and basal dendritic zones of fields CA₁ and CA₃ the overwhelming majority of the synapses are of the asymmetrical variety, the axon terminals ending principally upon dendritic spines, and to a lesser extent upon the shafts and secondary or tertiary branches of the dendrites. Between 1 and 8% of the axon terminals in these zones contained flattened vesicles: all of these formed symmetrical contacts upon medium-sized or large dendritic shafts. In the molecular layer of the dentate gyrus a slightly higher percentage of flattened vesicle containing profiles was observed (10%); again these formed symmetrical contacts upon dendritic shafts. In the stratum pyramidale of the hippocampal fields and the stratum granulosum of the dentate gyrus of the rat, flattened vesicle containing synapses are two or three times more numerous than those with spheroidal vesicles. In the cat hippocampus the axosomatic synapses are about equally distributed between those containing round, and those with flattened vesicles.The finding that at the focus of post-synaptic inhibition, at the level of the pyramidal cell somata, the majority of the axon terminals contains flattened synaptic vesicles, whereas in the region of termination of the extrinsic, commissural and long association pathways (all of which are excitatory) virtually all the synapses contain round vesicles, strongly supports the view that endings containing flattened vesicles mediate post-synaptic inhibition in the hippocampal formation.Supported in part by Grant EY-00599 from the National Eye Institute.We should like to thank Mr. Paul Myers and Mr. Milburn W. Rhoades for their technical assistance, and Mrs. Doris Stevenson for secretarial help.     

Transition vesicle-enriched fractions isolated from rat liver exhibit a unique peptide pattern

Summary Isolated fractions enriched in transition vesicles isolated by preparative free-flow electrophoresis from transition elements of endoplasmic reticulum incubated with ATP and a cytosol fraction in a cell free system, exhibited a polypeptide composition distinct from that of the original transitional endoplasmic reticulum fractions. The transition vesicles were deficient in peptides of apparent molecular mass between 200 and 270 kD, in the vicinity of 65 kD and less than 40 kD. Immunoblot analysis suggested a 140 kD protein to be concentrated in transition vesicles and low or absent from the endoplasmic reticulum fractions from which the transition vesicles were derived.     

Models of active transport of neurotransmitters in synaptic vesicles

Models of the active transport of neurotransmitters in synaptic vesicles were constructed. The models were used to determine the resting potential at membranes of synaptic vesicles: 40mV (monoamines and acetylcholine) and -40mV (glutamate). The potential at the membrane of a synaptic vesicle was almost absent for the transport of GABA and glycine. The neurotransmitter concentration of a cell was 0.1-18mM at the concentration of neurotransmitters in a vesicle equal to 0.5M. This result is in qualitative agreement with the relevant experimental data.     

Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury

Oxidative stress, an imbalance between oxidants and antioxidants, contributes to the pathogenesis of traumatic brain injury (TBI). Oxidative neurodegeneration is a key mediator of exacerbated morphological responses and deficits in behavioral recoveries. The present study assessed early hippocampal sequential imbalance to possibly enhance antioxidant therapy. Young adult male Sprague-Dawley rats were subjected to a unilateral moderate cortical contusion. At various times post-TBI, animals were killed and the hippocampus was analyzed for antioxidants (GSH, GSSG, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, superoxide dismutase, and catalase) and oxidants (acrolein, 4-hydroxynonenal, protein carbonyl, and 3-nitrotyrosine). Synaptic markers (synapsin I, postsynaptic density protein 95, synapse-associated protein 97, growth-associated protein 43) were also analyzed. All values were compared with those for sham-operated animals. Significant time-dependent changes in antioxidants were observed as early as 3 h posttrauma and paralleled increases in oxidants (4-hydroxynonenal, acrolein, and protein carbonyl), with peak values obtained at 24-48 h. Time-dependent changes in synaptic proteins (synapsin I, postsynaptic density protein 95, and synapse-associated protein 97) occurred well after levels of oxidants peaked. These results indicate that depletion of antioxidant systems following trauma could adversely affect synaptic function and plasticity. Early onset of oxidative stress suggests that the initial therapeutic window following TBI appears to be relatively short, and it may be necessary to stagger selective types of antioxidant therapy to target specific oxidative components.     

Identification of intrinsic sialidase and sialoglycoprotein substrates in rat brain synaptic junctions

Sialidase activity associated with rat brain synaptic junctions (SJ) and synaptic membranes (SM) was determined. Both fractions released sialic acid from exogenous glycopeptides and gangliosides. SJ accounted for 5-10% of the total sialidase activity recovered from SM following extraction with Triton X-100, and the specific activity of SJ sialidase was 60% of that of the parent SM fraction. Intrinsic SJ sialidase hydrolysed 12-15% of the sialic acid associated with endogenous SJ glycoproteins. Sialic acid residues associated with SJ glycoproteins were labelled with sodium borotritide and SJ proteins fractionated by affinity chromatography on concanavalin A-agarose. SJ glycoproteins that reacted with concanavalin A (con A+ glycoproteins) accounted for 25% of the total SJ [3H]sialic acid. Intrinsic SJ sialidase hydrolysed 20% of the [3H]sialic acid associated with these glycoproteins. Each molecular weight class of con A+ glycoprotein previously shown to be a specific component of the postsynaptic apparatus contained sialic acid and was acted on by intrinsic SJ sialidase.     

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)     

O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.     

Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.     

The duration of aldehyde fixation as a “flattening factor” of synaptic vesicles

Summary Synaptic vesicle flattening can be induced in the excitatory mossy fibre endings of the rat cerebellum by prolonged immersion in aldehyde during fixation (with or without perfusion). The flattening is found in a greater percentage of vesicles if perfusion has been omitted before the prolonged immersion. This is discussed in relation to the various other factors that are thought to cause flattening and the important problem of the classification of different types of synapse.The author wishes to express his thanks to Prof. E.G. Gray, for advice and constant help; to Mrs. H. Samson and M. Lourdes Brito for technical assistance, and to the Instituto de Alta Cultura, Lisbon, for a grant.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.     

Plasma membrane dehydrogenases in rat brain synaptic membranes. Multiplicity and subunit composition

Plasma membrane redox enzymes have been investigated in synaptic membranes from rat brain nerve terminals. UV-Vis spectra of intact synaptic plasma membranes are presented and the presence of ab-type cytochrome, detectable at 77°K and sensitive to NADH or NADPH, is shown. The molecular characterization of rat synaptic NADH-dehydrogenases was further performed on solubilized enzymes using a recently developed nondissociating polyacrylamide gel electrophoresis technique. Synaptic plasma membranes were solubilized with 1% sodium cholate or Triton X-114 and centrifuged. The supernatant retained over 60% of the NADH-dehydrogenase activity, tested with either DCIP or ferricyanide as substrates, together with NADH. Both enzyme activities were insensitive toward rotenone. This extraction procedure also solubilized about 50% of the proteins. When submitted to polyacrylamide gel electrophoresis under nondenaturing conditions and stained for NADH-dehydrogenase activity, five bands of different mobilities were detected. The multiple NADH-dehydrogenases of synaptic plasma membranes were investigated by means of band excision and the five excised bands each submitted to amino acid analysis and to 2-D electrophoresis. The subunit composition of each band was then deduced, together with the molecular weight and pI of each respective subunit. NADH-dehydrogenases have also been purified by means of FPLC on Mono-P (chromatofocusing) followed by gel filtration on Superose 12. NADH-Dehydrogenase IV and V could be purified in their active forms by this approach.Abbreviations DCIP dichlorophenol-indophenol - FPLC fast protein liquid chromatography.