Electrokinetic properties of isolated cerebral-cortex synaptic vesicles

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.     

Exocytotic steps in cell-free system after cholesterol deprivation in synaptosomal plasma membranes and synaptic vesicles

Using a cell-free system we investigated a specific role of cholesterol in exocytotic processes. To modulate the cholesterol content in membrane methyl-beta-cyclodextrin was used as a cholesterol binding agent. The experimental conditions for cholesterol depletion from synaptosomal membrane structures were determined and depended on methyl-beta-cyclodextrin concentration, time and mediums temperature. The role of cholesterol was studied on the stages of synaptic vesicles docking and Ca(2+)-stimulated fusion which are the components of multivesicular compound exocytosis. Using dynamic light scattering technique we have found that after cholesterol depletion from synaptic vesicles the process of their aggregation (docking) remains unchanged. It was found that the rate of calcium-triggered fusion of synaptic vesicles depends on the membrane level of cholesterol. The decreasing level of synaptosomal plasma membrane cholesterol by 8% leads to suppression of the Ca(2+)-dependent membrane fusion with synaptic vesicles. But, under 25% reduction of plasma membrane cholesterol the level of membrane merging with synaptic vesicles did not differ from control; probably this is due to changes in physical properties of lipid bilayer and/ or disturbances in function of membrane proteins driving this process. In cholesterol depleted synaptosomes the exocytotic release of glutamate stimulated by calcium was decreased by 32%. Obtained data suggest that the cholesterol concenration in synaptosomal plasma membranes or synaptic vesicles is the crucial determinant for synaptic transmission efficiency in nerve terminals.     

Calcium-triggered fusion of synaptic vesicles and synaptosomal plasma membranesin vitro

We have developed a system, in which fusion of synaptic vesicles with synaptosomal plasma membranes in the presence of synaptic soluble proteins can be studiedin vitro. We found that in this system micromolar concentrations of Ca²⁺ trigger fusion. The extent of fusion is insensitive to Ca²⁺ in millimolar concentrations, but can be covered by addition of MgATP. Ultimately, characterization of such cell-free systems makes it possible to identify biochemical events, which mediate and regulate these membrane fusion eventsin vivo.     

Creatine kinase isoenzyme associated with synaptosomal membrane and synaptic vesicles

Brain creatine kinase is principally of soluble cytoplasmic origin (anodal electrophoretic mobility). However, synaptosomal membranes and synaptic vesicles are enriched in an isoenzyme electrophoretically similar to muscle type creatine kinase (cathodal electrophoretic mobility), but which can be distinguished from muscle type by other means.     

Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions.

Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamide gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain a high Km and a low Km protein kinase activity while only the high Km activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.     

Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5'-nucleotidase, Na+, K+-activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000--33000 are found. A gel-electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr 160000, 147000, 34000 and 25000) was made. Comigration of major bands was detected in one-dimensional gel electrophoresis with the 42000-Mr, 35000--33000-Mr and 34000-Mr components. Upon two-dimensional gel electrophoresis the 42000-Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin-like polypeptides is unlikely. Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.     

Electrokinetic behavior of inside-out vesicles from human red cell membranes

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (<5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.     

Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions

Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamine gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain at high K_m and a low K_m protein kinase activity while only the high K_m activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.     

Separation of enriched synaptosomes, synaptic vesicles and synaptic plasma membranes]

A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy     

ATP-dependent fusion of synaptic vesicles and synaptosomal plasma membrane in a cell-free system

The final step in exocytosis is the fusion of synaptic vesicle membrane with the synaptosomal plasma membrane, leading to the release of the neurotransmitters. We have reconstituted this fusion event in vitro, using isolated synaptic vesicles and synaptosomal plasma membranes from the bovine brain. The membranes of synaptic vesicles were loaded with the lipid--soluble fluorescent probe octadecylrhodamine B at the concentration that resulted in self-quenching of its fluorescence. The vesicles were then incubated with synaptosomal plasma membranes at 37 degrees C and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Synaptic vesicles by themselves did not fused with plasma membrane, only addition of ATP induced the fusion. W-7 and trifluoroperasine, the drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the ATP-induced fusion synaptic vesicles and synaptosomal plasma membranes. Our results indicate that the membrane fusion in the nerve terminals during exocytosis may be under direct control of calmodulin-dependent protein phosphorylation.     

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.     

Protein organization of rat synaptic plasma membranes and synaptic vesicles: A one- and two-dimensional study

The protein organization of rat brain synaptic plasma membranes (SPM) and synaptic vesicles (SV) was investigated by surface iodination and one- and two-dimensional electrophoresis. Polypeptides of molecular weights (MWs, in Kilodaltons) 170 K, 135 K, 96-86 K, 68-64-61 K, 56 K, 52 K, 38 K, 35-33 K, and 18 K are predominantly or exclusively exposed on the extracellular side of synaptosomes. Several polypeptides of MW between 70 K and 40 K are exclusively exposed on the cytoplasmic side of SPM. The use of two-dimensional electrophoresis allowed to recognize that, for some classes of MW, there are polypeptides of nearly the same MW and different isoelectric points exposed on both sides of SPM. The synaptosomal membrane shows a predominance of acidic proteins on the extracellular side and more neutral and basic proteins on the cytoplasmic side. With respect to SPM, SV are particularly enriched with polypeptides of MW 71 K, 56 K, 39-38 K, 32 K, 16 K, and 15 K. One of them, a doublet of MW 39-38 K, is the most highly labeled species upon surface iodination and is similar, but not identical, with a doublet located on the cytoplasmic side of SPM.     

Preparation and properties of vesicles enclosed by fatty acid membranes.

Stable preparations of microscopic particles were obtained from long-chain fatty acids by mechanical agitation of evaporated films in presence of buffer solutions. Oleic and linoleic acids were used. Studies of osmotic swelling and shrinking of the particles indicated that they are enclosed by semipermeable membranes. The particles, which were named ufasomes, are also capable of entrapping glucose in spaces inaccessible to enzymes. It was concluded that the ufasomes closely resemble phospholipid liposomes in their structure and properties.     

Dolichol alters dynamic and static properties of mouse synaptosomal plasma membranes

Dolichols are isoprenologues that are found in almost all tissues and whose biochemical function, aside from dolichol phosphate precursors, is not known. In addition, an understanding of the organizational and dynamic properties of dolichols in biological membranes has not been forthcoming. The purpose of the experiments reported here were to examine the effects of dolichol on the physical properties of mouse synaptic plasma membranes (SPM). Differential polarized phase fluorometry indicated that dolichol both fluidized and rigidified SPM. Membrane areas detected by diphenylhexatriene and trans-parinaric acid were selectively fluidized and rigidified, respectively. It also was found that the spin label, 5-doxyl stearic acid indicated that dolichol reduced membrane fluidity. These results report for the first time a structural effect of dolichol on a biological membrane.     

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Localization in the synaptic junction of the cyclic AMP stimulated intrinsic protein kinase activity of synaptosomal plasma membranes.

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggests that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Electrokinetic isolation of vesicles and ribosomes derived from Serratia marcescens.

Ribosomes and vesicles derived from the bacterium Serratia marcescens were separated from each other and from solubles using density gradient electrophoresis. Transport relationships were used to determine the electrophoretic mobilities of the particles. The effects of convection, sedimentation and diffusion were found to be negligible. The electrophoretic mobility obtained for the ribosome peak is -7 x 10(-5) cm2/(V.s). Under appropriate conditions, two vesicles peaks were obtained, the first with a mobility of -4 x 10(-5) cm2/(V.s) and the second with -9 x 10(-6) cm2/(V.s). This information can be used to predict the resolution of the separands in large-scale electrophoretic separations.