Journal of Neuroscience Research 3: 103-1 13 (1977)
Membrane Protein and Glycoprotein Composition of Beef Brain Synaptic Vesicles Christiane Richter-Landsberg, Volker Neuhoff, and Thomas V. Waehneldt Max-Planck-institute fur experimen telle Medizin, Forschungsstelle Neurochemie, Gottingen, West Germany
Synaptic vesicles were isolated from adult bovine cortical gray matter by differential centrifugation and membrane filtration of a hypoosmotically lysed crude mitochondria1 fraction. Vesicle preparations were analyzed for purity by electron microscopy and enzyme assays. Polyacrylamide gel electrophoresis of SDS-solubilized and 2-mercaptoethanol-reduced vesicle membrane proteins revealed 4 major proteins with molecular weights ranging from 17,000 t o 60,000, and about 10 minor proteins with molecular weights up t o 170,000. The protein profile of the Triton X-1 00-extracted vesicle membranes was less complex, with 1 major protein and 5 minor bands. The major protein of the Triton extract was identified as a glycoprotein with a molecular weight of 45,000. Two additional minor PASpositive bands were seen, with molecular weights of 78,000 and 95,000. Key words: synaptic vesicles, membrane proteins, glycoproteins, beef brain, SDS-electrophoresis
INTRODUCTION
In recent years the polyacrylamide gel electrophoresis in SDS-containing buffer systems has become a powerful tool for the characterization of water-insoluble membrane proteins (Maizel, 197 1;Waehneldt and Neuhoff, 1972). There is increasing evidence that membranes o f defined origin have only a limited number of intrinsic protein components (Waehneldt and Neuhoff, 1974; Waehneldt et al., 1973). Therefore, in subcellular fractionations pure fractions exhibit a simplified protein profile upon electrophoresis as contrasted with the starting material. I n the present paper this technique has been applied t o the analysis of the membrane proteins of synaptic vesicles. Since theoretically the proteins of synaptic vesicles constitute only 0.2 mg/g o f total rat brain tissue (Morgan et al., 1973a), it appears advisable to use gray matter from a readily available animal with a large brain. I n these Abbreviations: SDS, sodium dodecyl sulfate; PAS, periodic acid-Schiff stain; EDTA, ethylenediaininetetraacetic acid. Address reprint requests t o Dr. C. Richter-Landsberg, Prof. Dr. V. Neuhoff, Dr. T.V. Waehneldt, MaxPlanck-Institut fur experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Str. 3 , D 3400 Gottingen, West Germany.
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experiments we used beef brain, applying a modified procedure that is based on the classical methods of Whittaker et al. (1964) and De Robertis et al. (1963), as modified by Morgan et al. (1973a). The vesicle preparations were characterized by electron microscopy and by enzyme assays, and the membrane-bound proteins were analyzed by gel electrophoresis in SDS buffer systems under nonreducing as well as reducing conditions. For further characterization synaptic vesicle membrane proteins were extracted with Triton X-100, a detergent known to extract glycoproteins rather selectively from nervous tissue (Brunngraber, 1970; Lapetina and De Robertis, 1968). As macromolecular components of the synaptic machinery (Burger, 1973; Hughes, 1973) glycoproteins may have important functions during transmitter release, particularly in the exocytosis-endocytosis cycle for which there is increasing experimental evidence (Breckenridge and Morgan, 1972; Zimmermann and Whttaker, 1974a; Zimmermann and Whittaker, 1974b;Morgan et al., 1973b). MATERIALS AND METHODS Animal and Preparation of Brain
Adult beef brains, obtained from a local slaughterhouse, were placed in ice-cold isotonic (0.32 M) sucrose and were brought to the laboratory. Within less than 1 hour after the death of the animals gray cortical matter-enriched material was scraped off in the cold room at 4°C. Unless stated otherwise, all other procedures were also carried out in the cold. Preparation of Synaptic Vesicles
In a typical experiment, 90 g tissue (commonly obtained from 2 brains) were homogenized in an Potter-type Teflon-glass homogenizer in a total of 1,800 ml 0.32 M sucrose, 0.1 M EDTA, 1 mM sodium phosphate, pH 7.5, with 10 up-and-down strokes and centrifuged at 1,000 g, for 10 minutes in the GS-3 rotor of the Sorvall RC2-B centrifuge. The cloudy supernatant was removed by suction, refilled to 1,800 ml with the homogenization medium, and centrifuged at 11,500 g,, for 20 minutes (GS-3 rotor). After discarding of the microsomal supernatant the crude mitochondria1 pellet was subjected to threefold washing in isotonic homogenization medium at 1 1,500 ,g, for 20 minutes. The pellet finally obtained was hypoosmotically shocked by vigorous homogenization in 0.1 mM EDTA, 1 mM sodium phosphate, pH 7.5 (4 ml/g fresh tissue) and centrifuged at 25,000 g, for 60 minutes in the 6 X 60 ml fixed angle rotor (Omega I1 ultracentrifuge, Heraeus Christ GmbH). The supernatant - removed by suction - was brought to a final concentration of 0.32 M sucrose by addition of 1.6 M sucrose (yielding a total of approximately 360 ml), then distributed among 6 tubes that were each overlayed with 10 ml water, and then centrifuged at 45,000 g, for 7 hours in two 3 X 75 ml swingout rotors (Omega I1 ultracentrifuge, Heraeus Christ GmbH). The supernatant was carefully removed by suction, care being taken to avoid the portions close to the top as well as t o the pellet, and was passed successively through Sartorius membrane filters of 450, 300, and 200 nm pore size by gentle suction. The faintly opalescent filtrate (approximately 300 ml) was diluted fourfold with 0.1 mM EDTA and 1 mM sodium phosphate, pH 7.5, and centrifuged overnight at 22,000 g,, (GSA rotor). The clear supernatant was carefully removed by suction and discarded, and the loose pellet was filled to 12 ml with 0.1 mM
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EDTA and 1 mM sodium phosphate, pH 7.5, and centrifuged at 100,000 ,g for 60 minutes (8 X 10 ml fixed angle rotor, Omega I1 ultracentrifuge) to give a dense and virtually clear pellet of synaptic vesicles. The preparation took 24 hours, and the average yield was 0.06 mg vesicle protein/g fresh tissue. EIect ron Microscopy
Pellets of synaptic vesicles were fixed in cold 2.5% glutaraldehyde and postfixed in cold 1% osmic acid. After dehydration with ethanol of increasing concentration and after embedding in Epon, thin sections were cut with an LKB ultratome I1 and doublestained with 1% uranyl acetate and 1.5% lead citrate. Sections were then examined in a Jeol 100 B electron microscope. Polyacrylamide Gel Electrophoresis
SDS electrophoresis was carried out at room temperature in the following discontinuous buffer system: upper buffer, 0.05 M Tris-glycine (pH 8.9) plus 0.1% SDS; lower buffer, 0.1 M Tris-C1 (pH 8.1); gel buffer, 0.372 M Tris-C1 (pH 8.9); in 12% acrylamide gels (6.5 cm), with a 4% acrylamide stacking gel (1 cm); diameter of the tubes was 5 mm (Waehneldt and Mandel, 1972). To each gel 50 mg of protein was subjected. Voltage applied was 100 V throughout the entire electrophoresis. Reduction of the protein samples was carried out with 1% 2-mercaptoethanol under nitrogen. The samples were then heated for I minute at 100°C. After cooling to room temperature a twofold molar excess of iodoacetamide over 2-mercaptoethanol was added, and the samples were incubated for 30 minutes at 37°C and dialyzed overnight against a large excess of 0.4% SDS (Richter-Landsberg et al., 1974). Staining for protein was done with 0.2% Coomassie brilliant blue R 250 in methano1:acetic acid:water (50: 10:40, by volume). Staining for glycoprotein was carried out according to Zacharius et al. (1969), with periodic acid and Schiff s reagent. Coomassie blue-stained gels were scanned with a Joyce Loebl microdensitometer with a filter of 620 nm. The extinction output was measured with an Aristo 1130 L planimeter. Extraction With Triton X-I 00
Samples were homogenized in 0.5% (wtlvol) Triton X-100 (1 mg protein/ml) and centrifuged at 100,000 ,g for 15 minutes. The clear supernatant was removed and dialyzed for 24 hours against 0.4% SDS. The Triton X-100-insoluble residue was washed twice with water. The pellet was then dissolved in 0.4% SDS. Both SDS extracts were subjected to SDS electrophoresis. Enzyme Assays and Protein Determination
Lactate dehydrogenase (EC 1.1.1.27), monoamine oxidase (EC 1.4.3.4.), acid phosphatase (EC 3.1.3.2.), acetylcholinesterase (EC 3.1.1.7), 5’-nucleotidase (EC 3.1.3.9, and (Na+ -K+)-ATPase (EC 3.6.1.3.) were assayed as described by Morgan et al. (197 1). 0-Galactosidase (EC 3.2.1.23) and 0-glucosidase (EC 3.2.1.21) were assayed by the method of Gatt and Rapport (1966), and alkaline phosphatase by that of Cotman and Matthews (1971). The protein content of samples was determined according to the procedure of Lowry et al. (195 1) with bovine serum albumin as a standard.
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RESULTS
Electron microscopy of filtered synaptic vesicles is shown in Fig. 1. Visual inspection reveals that contamination with membrane profiles other than synaptic vesicles amounts to approximately 20-30%; these large vesicular fragments most likely constitute synaptosomal plasma membranes. Their occurrence has been substantially reduced by successive filtration steps (see Materials and Methods). Prior to filtration only about half of the membrane profiles are of the size of synaptic vesicles (not shown here). Myelin fragments and mitochondria are completely absent either before or after filtration. Enzymic characterization of synaptic vesicle preparations are given in Table I. For comparison, enzyme activities found in the total homogenate of bovine gray matter are also shown. The values for lactate dehydrogenase demonstrate that contamination with soluble proteins is low when compared with the total homogenate and even lower when compared with the soluble protein fraction in which lactate dehydrogenase is located (see Morgan et al., 1973a). The same applies to Contamination with outer mitochondria1 menibranes and lysosomes because of the absence of activity of monoamine oxidase and low values of 0-glucosidase, 0-galactosidase, and acid phosphatase, respectively. Other enzyme markers were used to test for the presence of plasma membrane fragments. Acetylcholinesterase and particularly alkaline phosphatase are low. The values of 5’-nucleotidase and especially Na+-K+-ATPase strongly point to the presence of synaptosomal plasma membranes; these contaminations persist - although at reduced level - even after passage through membrane filters, pointing to the large vesicular fragments seen in electron microscopical ex-
Fig. 1. Field of synaptic vesicle preparation (Magnification x 34,000). Besides some larger vesicular bodies most of the structures are of the size of synaptic vesicles.
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amination (Fig. 1). SDS gel electrophoretic profiles and corresponding scans of nonreduced and reduced synaptic vesicle proteins are shown in Fig. 2 and in Fig. 3, respectively. Molecular weights were determined by admixing marker proteins as described TABLE I. Assessment of Contamination in Synaptic Vesicle Fraction* Enzyme Lactate dehydrogenase Acid phosphatase p-Gahctosidase p-Glucosidase (Na+-K+)-ATPase 5 '-Nucleotidase Alkaline phosphatase Acetylcholinesterase Monoamine oxidase
Total homogenate
30.0 15.6 0.06 0.09 13.2 1.5 9.0 1.8 0.016
Synaptic vesicles
2.1 0.3 0.018 0.04 1.8 0.6 0.18 0.6 n.d.t
In reference fractions 144.8 150.0 0.24 0.3 103.1 1.2
soluble proteins lysosome enriched fraction
1
synaptosomal
8.5 0.18 mitochondria
*Values are means of specific enzymatic activities from 4 preparations (pmoles subtrate consumed/mg protein per hour). ? N o t detectable.
Fig. 2. Polyacrylamide gel electrophoresis of SDS-extracted synaptic vesicle fraction from ox brain (12% acrylamide gels, stained with Coomassie brilliant blue): 1) profile of unreduced synaptic vesicle proteins; 2) synaptic vesicle proteins reduced with 1%2-mercaptoethanol; 3) synaptic vesicle proteins reduced with 1% 2-mercaptoethanol and carboxymethylated with iodoacetamide (see Materials and Methods); 4) synaptic vesicle proteins same as sample 3 , mixed with reduced marker proteins (a, Transferrin; b, Ovalbumin; c, Cytochrome C).
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Fig. 3. Densitometric tracings of fractionated vesicle proteins in discontinuous SDS buffer system. (same samples as in Fig. 2). The electrophoretic profiles were used for determination both of molecular weights and percentage distribution of individual proteins (see also Table 11).
by Weber and Osborn (1969) (transferrin, 90,000; ovalbumin, 45,000; Cytochrome C , 12,100). The molecular weights of the vesicle proteins, their logarithms being inversely related t o the rate of anodic migration, are listed in Table 11. According to Weber and Osborn (1969) the molecular weight determination is reliable in the range of 10%. Also added are values of relative contributions of single protein bands, expressed as percentage of the total protein content. It is evident (Fig,2 and 3, Table 11) that the membrane proteins of synaptic vesicles display substantial complexity. In the nonreduced sample 5-6 major protein bands are visible besides approximately 15 minor protein bands. Among the major proteins peak I, 11, and I11 are most prominent, having molecular weights of 60,000,45,000, and 38,000 respectively. Upon reduction with 2-mercaptoethano1, followed by carboxymethylation, the protein profile is more simplified. Among others, 4 major bands are listed in Table 11, among which bands I, 11, 111, and 19 show substantial increase relative to the unreduced sample. A corresponding decrease of protein bands, particularly in the high-molecularweight region (80,000) is noticeable. This result indicates that part of the membrane proteins of synaptic vesicles have disulfide bridges when treated with SDS under nonreducing conditions. Bands 17 and 18 (Table 11) disappear upon reduction, excluding the POSsibility of myelin contamination, since myelin basic protein and myelin proteolipid protein (Waehneldt and Neuhoff, 1972) remain unchanged under reductive treatment as employed in this communication, i.e., maximally 1% 2-mercaptoethanol (Richter-Landsberg, unpublished).
*
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TABLE 11. Membrane Proteins of Synaptic Vesicles Prior to reduction No.
MW
Distribution
1-6
170,000115,000 109,000 96,000 79,000 67,000 60,000 49,000 45,000 42,000 39,000 38,000 34,000 33,000 29,000 23,000 19,500 17,500 14,500
18.0
7 8 9 10 1 11 I1 12 13 I1 I 14 15 16 17 18 19 20
6 .O 5.9 7.5 5 .O 13.4 12.9
9.1 9.2 7.9 1.0 3.7 1.5
__ After reduction MW Distribution
170,0001 15,000 110,000 93,000 82,000 -
60,000 49,000 45,000 42,000 39,000 38,000 33,000 29,000 27,000 -
7.8 3.2 3.8 3.9 ~
19.8 16.9
12.2 18.0 -
-
-
17,500 14,500
10.1 4.0
*Molecular weights and distribution, expressed as percentage of total protein (determined in 12% polyacrylamide gels).
For further characterization synaptic vesicle membranes were extracted with Triton X-100. This detergent tends to extract a specific set of proteins, among which glycoproteins are predominant. Despite repeated extraction with Triton X-100 only 35% of the synaptic vesicle proteins were extractable; the insoluble residue was rendered soluble only by the attack of SDS. This is in contrast to the synaptosomal plasma membrane of which Triton X-100 solubilizes about 70% of total protein (Waehneldt et al., 1971). The Triton extract of synaptic vesicles was electrophoresed after dialysis against 0.4% SDS, as well as the SDS-solubilized residue of the Triton extract. Pherograms and molecular weights are shown in Fig. 4 and Table 111. The synaptic vesicle proteins extractable with Triton X-100 consist of 1 major protein (MW 45,000), which comprises about 25% of all protein-staining material (band 11, Table HI), and of 5 minor proteins. Upon staining for glycoprotein with periodic acid-Schiff reagent, only 3 bands were found (Table 111), of which band I1 is predominant, whereas 2 high-molecular-weight bands are minor. Attempts to change the protein or glycoprotein profile of the Triton extract by reduction were unsuccessful. Preliminary results indicate that the major synaptic vesicular glycoprotein (band 11) is acidic (Richter-Landsberg, unpublished).
DISCUSSION
In the present work attempts are described t o isolate synaptic vesicles from beef brain. Beef was chosen because of its availability and because of its large brain, which permits the bulk enrichment of forebrain gray matter. The preparative method used is
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Fig. 4. Densitometric tracing of fractionated synaptic vesicle proteins after extraction with Triton X-100 in discontinuous SDS buffer system : upper tracing) fractionation of whole synaptic vesicle proteins; middle tracing) fractionation of the Triton X-100-insoluble synaptic vesicle proteins; lower tracing) fractionation of the Triton X-100-soluble synaptic vesicle proteins after dialysis against 0.4% SDS solution (see also Table 111).
TABLE 111. Triton X-100-extractable Membrane Proteins of Synaptic Vesicles* No.
Protein stain with Coomassie blue
Glycoprotein stain with ueriodic acid-Schiff rearent
1 2 I1 3 4 5
95,000 78,000 45,000 42,000 27,000 17,500
95,000 78,000 45,000 -
-
*Molecular weights (determined in 12% polyacrylamide gels)
based on earlier work (Morgan et al., 1973a; Whittaker et al., 1964; De Robertis et al., 1963), i.e., isolation of a nucleusfree and microsomefree crude mitochondrial fraction. In contrast to these workers no step was included to isolate synaptosomes, since synaptic vesicles have been shown to display extraordinarily low densities upon sucrose gradient centrifugation, permitting separation from heavier subcellular particles. Instead, the crude mitochondrial fraction was hypoosmotically shocked, thus releasing synaptic vesicles that were then separated from the bulk of other suborganelles by differential centrifugation in low-density sucrose. Finally, the crude vesicle fraction obtained was then substantially freed from large vesicular membrane fragments by passage through filters of decreasing pore size, a method that has been successfully applied by Morgan et al. (1973a) for total rat brain synaptic vesicles.
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The synaptic vesicle preparations of this paper were studied by electron microscopy and by enzyme assays. The results indicate that our fractions are about 70% pure, which is in agreement with the purity of synaptic vesicle fractions from guinea pig and rat brain as described by other workers(Morgan et al., 1973a;De Robertis et al., 1963; Soller et al., 1973). Electrophoretic analysis confirms that the synaptic vesicles have reduced complexity in their protein profile as contrasted with the membrane fraction of the total homogenate. Nevertheless, the protein profiles are rather complex even after reductive treatment, with 4 major protein bands each occupying 12-20% of total proteins. These high values suggest that the major proteins are intrinsic synaptic vesicle components. The origins of the minor protein components are difficult to assess, since they may be either of synaptic vesicular or of any other nature. The data of enzyme analyses point to contamination with synaptosomal plasma membranes because of elevated levels of (Na+-K+)ATPase. The results of our electrophoretic analyses are similar to the results of Morgan et al. (1973b). They found about 7 major proteins with molecular weight ranges similar to those of our major components. They, too, describe the existence of 3 glycoprotein bands, which had, however, molecular weights up to 120,000, in contrast to 95,000 in our preparation. On the one hand these differences may be due to abnormal behavior of glycoproteins upon SDS gel electrophotesis (Bretscher, 1973). On the other hand our own results (not published) indicate that synaptic vesicles prepared by our method from beef as well as from rat brain have membrane proteins that are very similar or are even identical. Thus, differences in protein patterns between the work of Morgan et al. (1973b) and our own work are probably related t o differences in preparation and degree of purity. The extraordinary heterogeneity of mammalian central nervous system (CNS) synaptic vesicles, which originates in both regional differences and in a sizable number of transmitters, has been a major difficulty in obtaining very pure preparations. This has become evident in our work as well as in others (Morgan et al., 1973a; Whittaker, 1973; Marchbanks, 1974). Initial anaiyses of synaptic vesicles from the electric organ of Torpedo (Whittaker et al., 1974) shows a different and somewhat simplified protein pattern as compared to mammalian CNS synaptic vesicles. However, this simplified protein pattern may possibly be due to separation conditions that are not optimal for fractionation of water-insoluble membrane proteins (Richter-Landsberg et al., 1974; Ruche1 et al., 1974). Nevertheless, the exclusively cholinergic nature of synaptic vesicles from Torpedo will in the long run be the most suitable material for detailed study of protein patterns in order to relate single proteins to functional roles in the exocytosis-endocytosis cycle during synaptic release of transmitters.
ACKNOWLEDGMENTS The authors wish to thank Mrs. A. Wolff for the preparation of electron microscopic photographs and Miss H. Fruh for excellent technical help. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 33). This work is part of the doctoral thesis of C. Richter-Landsberg, Gottingen, 1975.
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REFERENCES Breckenridge, W.C., and Morgan, I.G. (1972). Common glykoproteins of synaptic vesicles and the synaptosomal plasma membrane. FEBS Lett. 22 :253-256. Bretscher, M.S. (1973). Membrane structure: Some general principles. Science 181:622--629. Brunngraber, E. (1970). Glycoproteins in neural tissues. In “Protein Metabolism of the Nervous System,” (A. Lajtha, ed.). New York-London: Plenum Press, pp. 383-407. Burger, M.M. (1973). The surface membrane and cell-cell interactions. In “The Neuroscience Third Study Program,’’ (F.O. Schmitt and F.G. Worden, eds.). Cambridge, Mass., London: MIT Press, pp 773782. Cotman, C.W., Matthews, D.H. (197 1). Synaptic plasma membranes from rat brain synaptosomes: Isolation and partial characterization. Biochim. Biophys. Acta 249:380-394. De Robertis, E., Arnaiz, G.R. de L., and Zieher, L.M. (1963). Isolation of synaptic vesicles and structural organization of the acetylcholine system within brain nerve endings. J . Neurochem. 10: 225-235. Gatt, S., and Rapport, M.M. (1966). Isolation of 0-galactosidase and P-glucosidase from brain. Biochim. Biophys. Acta 113567-576. Hughes, R.L. (1973). Glycoproteins as components of cellular membranes. Prog. Biophys. Mol. Biol. 26: 189-260. Lapetina, E.G., and De Robertis, E. (1968). Action of Triton X-100 on lipids and proteolipidsofnerveending membranes. Life Sci. 7:203-208. Lowry, O.H., Rosebrough, H.J., F a n , A.L., and Randall, R.J. (1951). Protein measurement with the fohn phenol reagent. J. Biol. Chem. 193 :265-275. Maize1 Jr., J.V. (197 1). Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5 : 179246. Marchbanks, R.M. (1974). Isolation and study of synaptic vesicles. Res. Methods in Neurochem. 2 : 79-97. Morgan, I.G., Wolfe, L.S., Mandel, P., and Gombos, G. (197 1). Isolation of plasma membranes from rat brain. Biochim. Biophys. Acta 24 1:737-75 1. Morgan, I.G., Vincendon, D., and Gombos, G. (1973a). Adult rat brain synaptic vesicles. 1. Isolation and characterization. Biochim. Biophys. Acta 320:671-680. Morgan, I.G., Breckenridge, W.C., Vincendon, G., and Gombos, G. (1973b). The proteins of nerve ending membranes. In “Proteins of the Nervous System.” New York: Raven Press, pp. 171L 192. Richter-Landsberg, C., Riichel, R., and Waehneldt, T.V. (1974). Discontinuous gel electrophoresis of reduced membrane proteins. Biochem. Biophys. Res. Comm. 59:781-788. Riichel, R ., Mesecke, S., Wolfrum, D.-I., and Neuhoff, V. (1974). Microelectrophoresis in continuouspolyacrylamide gradient gels. 11. Hoppe-Seyler’s 2. Physiol. Chem. 355:997-1020. Soller, M., Koenig, H., Mylroie, K., Hughes, C., and Lu, C.Y. (1973). Isolation and characterization of soluble acidic lipoproteins from rat brain synaptic vesicles. J. Neurochem. 21 557-572. Waehneldt, T.V., and Mandel, P. (1972). Isolation of rat brain myelin, monitored by polyacrylamide gel electrophoresis of dodecyl sulfateextracted proteins. Brain Res. 40:419-436. Waehneldt, T.V., Morgan, I.G., and Gombos, G. (1971). The synaptosomal plasma membranes protein and glycoprotein composition. Brain Res. 34:403-406. Waehneldt, T.V., and Neuhoff, V. (1972). Membrane proteins of the nervous system. Natunvissenschaften 5 9 :232 -239. Waehneldt, T.V., and Neuhoff, V. (1974). Membrane proteins of rat brain: Compositional changes during postnatal development. J. Neurochem. 23:71-77. Waehneldt, T.V., Ralston, H.J., and Shooter, E.M. (1973). The protein composition of the subcellular fractions from mouse brain. Neurobiology 3 :215-224. Weber, K., and Osborn, M. (1969). The reliability of molecular weight determination by dodecylsulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-44 12. Whittaker, V.P. (1973). The biochemistry of synaptic transmission. Natunvissenschaften 60:79-97. Whittaker, V.P., Michaelson, I.A., and Kirkland, R.J.A. (1964). The separation of synaptic vesicles from nerve-ending part icles (sy nap tosom es). Bio chem. J. 9 0 :29 3 -3 03.
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Whittaker, V.P., Dowdall, M.J., Dowe, G.H.C., Facino, R.M., and Scotto, J. (1974). Proteins of cholinergic synaptic vesicles from the electric organ of Torpedo : Characterization of a low molecular weight acidic protein. Brain Res. 75:115-131. Zacharius, R.M., Zell, T.E., Morrison, J.H., and Woodlock, J.J. (1969). Glycoprotein staining following electrophoresis on acrylamide gel. Anal. Biochem. 30:148-152. Zimmermann, H., and Whittaker, V.P. (1974a). Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapsis of the electric organ of Torpedo: A combined biochemical, electrophysiological and morphological study. J. Neurochem. 22 :435450. Zimmermann, H., and Whittaker, V.P. (1974b). Different recovery rates of the electrophysiological, biochemical and morphological parameters in the cholinergic synapses of the Torpedo electric organ after stimulation. J. Neurochem. 22:1109-1114.
Membrane Protein and Glycoprotein Composition of Beef Brain Synaptic Vesicles Christiane Richter-Landsberg, Volker Neuhoff, and Thomas V. Waehneldt Max-Planck-institute fur experimen telle Medizin, Forschungsstelle Neurochemie, Gottingen, West Germany
Synaptic vesicles were isolated from adult bovine cortical gray matter by differential centrifugation and membrane filtration of a hypoosmotically lysed crude mitochondria1 fraction. Vesicle preparations were analyzed for purity by electron microscopy and enzyme assays. Polyacrylamide gel electrophoresis of SDS-solubilized and 2-mercaptoethanol-reduced vesicle membrane proteins revealed 4 major proteins with molecular weights ranging from 17,000 t o 60,000, and about 10 minor proteins with molecular weights up t o 170,000. The protein profile of the Triton X-1 00-extracted vesicle membranes was less complex, with 1 major protein and 5 minor bands. The major protein of the Triton extract was identified as a glycoprotein with a molecular weight of 45,000. Two additional minor PASpositive bands were seen, with molecular weights of 78,000 and 95,000. Key words: synaptic vesicles, membrane proteins, glycoproteins, beef brain, SDS-electrophoresis
INTRODUCTION
In recent years the polyacrylamide gel electrophoresis in SDS-containing buffer systems has become a powerful tool for the characterization of water-insoluble membrane proteins (Maizel, 197 1;Waehneldt and Neuhoff, 1972). There is increasing evidence that membranes o f defined origin have only a limited number of intrinsic protein components (Waehneldt and Neuhoff, 1974; Waehneldt et al., 1973). Therefore, in subcellular fractionations pure fractions exhibit a simplified protein profile upon electrophoresis as contrasted with the starting material. I n the present paper this technique has been applied t o the analysis of the membrane proteins of synaptic vesicles. Since theoretically the proteins of synaptic vesicles constitute only 0.2 mg/g o f total rat brain tissue (Morgan et al., 1973a), it appears advisable to use gray matter from a readily available animal with a large brain. I n these Abbreviations: SDS, sodium dodecyl sulfate; PAS, periodic acid-Schiff stain; EDTA, ethylenediaininetetraacetic acid. Address reprint requests t o Dr. C. Richter-Landsberg, Prof. Dr. V. Neuhoff, Dr. T.V. Waehneldt, MaxPlanck-Institut fur experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Str. 3 , D 3400 Gottingen, West Germany.
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150 Fifth Avenue, New York, NY 10011
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Richter-Landsberg, Neuhoff, and Waehneldt
experiments we used beef brain, applying a modified procedure that is based on the classical methods of Whittaker et al. (1964) and De Robertis et al. (1963), as modified by Morgan et al. (1973a). The vesicle preparations were characterized by electron microscopy and by enzyme assays, and the membrane-bound proteins were analyzed by gel electrophoresis in SDS buffer systems under nonreducing as well as reducing conditions. For further characterization synaptic vesicle membrane proteins were extracted with Triton X-100, a detergent known to extract glycoproteins rather selectively from nervous tissue (Brunngraber, 1970; Lapetina and De Robertis, 1968). As macromolecular components of the synaptic machinery (Burger, 1973; Hughes, 1973) glycoproteins may have important functions during transmitter release, particularly in the exocytosis-endocytosis cycle for which there is increasing experimental evidence (Breckenridge and Morgan, 1972; Zimmermann and Whttaker, 1974a; Zimmermann and Whittaker, 1974b;Morgan et al., 1973b). MATERIALS AND METHODS Animal and Preparation of Brain
Adult beef brains, obtained from a local slaughterhouse, were placed in ice-cold isotonic (0.32 M) sucrose and were brought to the laboratory. Within less than 1 hour after the death of the animals gray cortical matter-enriched material was scraped off in the cold room at 4°C. Unless stated otherwise, all other procedures were also carried out in the cold. Preparation of Synaptic Vesicles
In a typical experiment, 90 g tissue (commonly obtained from 2 brains) were homogenized in an Potter-type Teflon-glass homogenizer in a total of 1,800 ml 0.32 M sucrose, 0.1 M EDTA, 1 mM sodium phosphate, pH 7.5, with 10 up-and-down strokes and centrifuged at 1,000 g, for 10 minutes in the GS-3 rotor of the Sorvall RC2-B centrifuge. The cloudy supernatant was removed by suction, refilled to 1,800 ml with the homogenization medium, and centrifuged at 11,500 g,, for 20 minutes (GS-3 rotor). After discarding of the microsomal supernatant the crude mitochondria1 pellet was subjected to threefold washing in isotonic homogenization medium at 1 1,500 ,g, for 20 minutes. The pellet finally obtained was hypoosmotically shocked by vigorous homogenization in 0.1 mM EDTA, 1 mM sodium phosphate, pH 7.5 (4 ml/g fresh tissue) and centrifuged at 25,000 g, for 60 minutes in the 6 X 60 ml fixed angle rotor (Omega I1 ultracentrifuge, Heraeus Christ GmbH). The supernatant - removed by suction - was brought to a final concentration of 0.32 M sucrose by addition of 1.6 M sucrose (yielding a total of approximately 360 ml), then distributed among 6 tubes that were each overlayed with 10 ml water, and then centrifuged at 45,000 g, for 7 hours in two 3 X 75 ml swingout rotors (Omega I1 ultracentrifuge, Heraeus Christ GmbH). The supernatant was carefully removed by suction, care being taken to avoid the portions close to the top as well as t o the pellet, and was passed successively through Sartorius membrane filters of 450, 300, and 200 nm pore size by gentle suction. The faintly opalescent filtrate (approximately 300 ml) was diluted fourfold with 0.1 mM EDTA and 1 mM sodium phosphate, pH 7.5, and centrifuged overnight at 22,000 g,, (GSA rotor). The clear supernatant was carefully removed by suction and discarded, and the loose pellet was filled to 12 ml with 0.1 mM
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EDTA and 1 mM sodium phosphate, pH 7.5, and centrifuged at 100,000 ,g for 60 minutes (8 X 10 ml fixed angle rotor, Omega I1 ultracentrifuge) to give a dense and virtually clear pellet of synaptic vesicles. The preparation took 24 hours, and the average yield was 0.06 mg vesicle protein/g fresh tissue. EIect ron Microscopy
Pellets of synaptic vesicles were fixed in cold 2.5% glutaraldehyde and postfixed in cold 1% osmic acid. After dehydration with ethanol of increasing concentration and after embedding in Epon, thin sections were cut with an LKB ultratome I1 and doublestained with 1% uranyl acetate and 1.5% lead citrate. Sections were then examined in a Jeol 100 B electron microscope. Polyacrylamide Gel Electrophoresis
SDS electrophoresis was carried out at room temperature in the following discontinuous buffer system: upper buffer, 0.05 M Tris-glycine (pH 8.9) plus 0.1% SDS; lower buffer, 0.1 M Tris-C1 (pH 8.1); gel buffer, 0.372 M Tris-C1 (pH 8.9); in 12% acrylamide gels (6.5 cm), with a 4% acrylamide stacking gel (1 cm); diameter of the tubes was 5 mm (Waehneldt and Mandel, 1972). To each gel 50 mg of protein was subjected. Voltage applied was 100 V throughout the entire electrophoresis. Reduction of the protein samples was carried out with 1% 2-mercaptoethanol under nitrogen. The samples were then heated for I minute at 100°C. After cooling to room temperature a twofold molar excess of iodoacetamide over 2-mercaptoethanol was added, and the samples were incubated for 30 minutes at 37°C and dialyzed overnight against a large excess of 0.4% SDS (Richter-Landsberg et al., 1974). Staining for protein was done with 0.2% Coomassie brilliant blue R 250 in methano1:acetic acid:water (50: 10:40, by volume). Staining for glycoprotein was carried out according to Zacharius et al. (1969), with periodic acid and Schiff s reagent. Coomassie blue-stained gels were scanned with a Joyce Loebl microdensitometer with a filter of 620 nm. The extinction output was measured with an Aristo 1130 L planimeter. Extraction With Triton X-I 00
Samples were homogenized in 0.5% (wtlvol) Triton X-100 (1 mg protein/ml) and centrifuged at 100,000 ,g for 15 minutes. The clear supernatant was removed and dialyzed for 24 hours against 0.4% SDS. The Triton X-100-insoluble residue was washed twice with water. The pellet was then dissolved in 0.4% SDS. Both SDS extracts were subjected to SDS electrophoresis. Enzyme Assays and Protein Determination
Lactate dehydrogenase (EC 1.1.1.27), monoamine oxidase (EC 1.4.3.4.), acid phosphatase (EC 3.1.3.2.), acetylcholinesterase (EC 3.1.1.7), 5’-nucleotidase (EC 3.1.3.9, and (Na+ -K+)-ATPase (EC 3.6.1.3.) were assayed as described by Morgan et al. (197 1). 0-Galactosidase (EC 3.2.1.23) and 0-glucosidase (EC 3.2.1.21) were assayed by the method of Gatt and Rapport (1966), and alkaline phosphatase by that of Cotman and Matthews (1971). The protein content of samples was determined according to the procedure of Lowry et al. (195 1) with bovine serum albumin as a standard.
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RESULTS
Electron microscopy of filtered synaptic vesicles is shown in Fig. 1. Visual inspection reveals that contamination with membrane profiles other than synaptic vesicles amounts to approximately 20-30%; these large vesicular fragments most likely constitute synaptosomal plasma membranes. Their occurrence has been substantially reduced by successive filtration steps (see Materials and Methods). Prior to filtration only about half of the membrane profiles are of the size of synaptic vesicles (not shown here). Myelin fragments and mitochondria are completely absent either before or after filtration. Enzymic characterization of synaptic vesicle preparations are given in Table I. For comparison, enzyme activities found in the total homogenate of bovine gray matter are also shown. The values for lactate dehydrogenase demonstrate that contamination with soluble proteins is low when compared with the total homogenate and even lower when compared with the soluble protein fraction in which lactate dehydrogenase is located (see Morgan et al., 1973a). The same applies to Contamination with outer mitochondria1 menibranes and lysosomes because of the absence of activity of monoamine oxidase and low values of 0-glucosidase, 0-galactosidase, and acid phosphatase, respectively. Other enzyme markers were used to test for the presence of plasma membrane fragments. Acetylcholinesterase and particularly alkaline phosphatase are low. The values of 5’-nucleotidase and especially Na+-K+-ATPase strongly point to the presence of synaptosomal plasma membranes; these contaminations persist - although at reduced level - even after passage through membrane filters, pointing to the large vesicular fragments seen in electron microscopical ex-
Fig. 1. Field of synaptic vesicle preparation (Magnification x 34,000). Besides some larger vesicular bodies most of the structures are of the size of synaptic vesicles.
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amination (Fig. 1). SDS gel electrophoretic profiles and corresponding scans of nonreduced and reduced synaptic vesicle proteins are shown in Fig. 2 and in Fig. 3, respectively. Molecular weights were determined by admixing marker proteins as described TABLE I. Assessment of Contamination in Synaptic Vesicle Fraction* Enzyme Lactate dehydrogenase Acid phosphatase p-Gahctosidase p-Glucosidase (Na+-K+)-ATPase 5 '-Nucleotidase Alkaline phosphatase Acetylcholinesterase Monoamine oxidase
Total homogenate
30.0 15.6 0.06 0.09 13.2 1.5 9.0 1.8 0.016
Synaptic vesicles
2.1 0.3 0.018 0.04 1.8 0.6 0.18 0.6 n.d.t
In reference fractions 144.8 150.0 0.24 0.3 103.1 1.2
soluble proteins lysosome enriched fraction
1
synaptosomal
8.5 0.18 mitochondria
*Values are means of specific enzymatic activities from 4 preparations (pmoles subtrate consumed/mg protein per hour). ? N o t detectable.
Fig. 2. Polyacrylamide gel electrophoresis of SDS-extracted synaptic vesicle fraction from ox brain (12% acrylamide gels, stained with Coomassie brilliant blue): 1) profile of unreduced synaptic vesicle proteins; 2) synaptic vesicle proteins reduced with 1%2-mercaptoethanol; 3) synaptic vesicle proteins reduced with 1% 2-mercaptoethanol and carboxymethylated with iodoacetamide (see Materials and Methods); 4) synaptic vesicle proteins same as sample 3 , mixed with reduced marker proteins (a, Transferrin; b, Ovalbumin; c, Cytochrome C).
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Fig. 3. Densitometric tracings of fractionated vesicle proteins in discontinuous SDS buffer system. (same samples as in Fig. 2). The electrophoretic profiles were used for determination both of molecular weights and percentage distribution of individual proteins (see also Table 11).
by Weber and Osborn (1969) (transferrin, 90,000; ovalbumin, 45,000; Cytochrome C , 12,100). The molecular weights of the vesicle proteins, their logarithms being inversely related t o the rate of anodic migration, are listed in Table 11. According to Weber and Osborn (1969) the molecular weight determination is reliable in the range of 10%. Also added are values of relative contributions of single protein bands, expressed as percentage of the total protein content. It is evident (Fig,2 and 3, Table 11) that the membrane proteins of synaptic vesicles display substantial complexity. In the nonreduced sample 5-6 major protein bands are visible besides approximately 15 minor protein bands. Among the major proteins peak I, 11, and I11 are most prominent, having molecular weights of 60,000,45,000, and 38,000 respectively. Upon reduction with 2-mercaptoethano1, followed by carboxymethylation, the protein profile is more simplified. Among others, 4 major bands are listed in Table 11, among which bands I, 11, 111, and 19 show substantial increase relative to the unreduced sample. A corresponding decrease of protein bands, particularly in the high-molecularweight region (80,000) is noticeable. This result indicates that part of the membrane proteins of synaptic vesicles have disulfide bridges when treated with SDS under nonreducing conditions. Bands 17 and 18 (Table 11) disappear upon reduction, excluding the POSsibility of myelin contamination, since myelin basic protein and myelin proteolipid protein (Waehneldt and Neuhoff, 1972) remain unchanged under reductive treatment as employed in this communication, i.e., maximally 1% 2-mercaptoethanol (Richter-Landsberg, unpublished).
*
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TABLE 11. Membrane Proteins of Synaptic Vesicles Prior to reduction No.
MW
Distribution
1-6
170,000115,000 109,000 96,000 79,000 67,000 60,000 49,000 45,000 42,000 39,000 38,000 34,000 33,000 29,000 23,000 19,500 17,500 14,500
18.0
7 8 9 10 1 11 I1 12 13 I1 I 14 15 16 17 18 19 20
6 .O 5.9 7.5 5 .O 13.4 12.9
9.1 9.2 7.9 1.0 3.7 1.5
__ After reduction MW Distribution
170,0001 15,000 110,000 93,000 82,000 -
60,000 49,000 45,000 42,000 39,000 38,000 33,000 29,000 27,000 -
7.8 3.2 3.8 3.9 ~
19.8 16.9
12.2 18.0 -
-
-
17,500 14,500
10.1 4.0
*Molecular weights and distribution, expressed as percentage of total protein (determined in 12% polyacrylamide gels).
For further characterization synaptic vesicle membranes were extracted with Triton X-100. This detergent tends to extract a specific set of proteins, among which glycoproteins are predominant. Despite repeated extraction with Triton X-100 only 35% of the synaptic vesicle proteins were extractable; the insoluble residue was rendered soluble only by the attack of SDS. This is in contrast to the synaptosomal plasma membrane of which Triton X-100 solubilizes about 70% of total protein (Waehneldt et al., 1971). The Triton extract of synaptic vesicles was electrophoresed after dialysis against 0.4% SDS, as well as the SDS-solubilized residue of the Triton extract. Pherograms and molecular weights are shown in Fig. 4 and Table 111. The synaptic vesicle proteins extractable with Triton X-100 consist of 1 major protein (MW 45,000), which comprises about 25% of all protein-staining material (band 11, Table HI), and of 5 minor proteins. Upon staining for glycoprotein with periodic acid-Schiff reagent, only 3 bands were found (Table 111), of which band I1 is predominant, whereas 2 high-molecular-weight bands are minor. Attempts to change the protein or glycoprotein profile of the Triton extract by reduction were unsuccessful. Preliminary results indicate that the major synaptic vesicular glycoprotein (band 11) is acidic (Richter-Landsberg, unpublished).
DISCUSSION
In the present work attempts are described t o isolate synaptic vesicles from beef brain. Beef was chosen because of its availability and because of its large brain, which permits the bulk enrichment of forebrain gray matter. The preparative method used is
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Fig. 4. Densitometric tracing of fractionated synaptic vesicle proteins after extraction with Triton X-100 in discontinuous SDS buffer system : upper tracing) fractionation of whole synaptic vesicle proteins; middle tracing) fractionation of the Triton X-100-insoluble synaptic vesicle proteins; lower tracing) fractionation of the Triton X-100-soluble synaptic vesicle proteins after dialysis against 0.4% SDS solution (see also Table 111).
TABLE 111. Triton X-100-extractable Membrane Proteins of Synaptic Vesicles* No.
Protein stain with Coomassie blue
Glycoprotein stain with ueriodic acid-Schiff rearent
1 2 I1 3 4 5
95,000 78,000 45,000 42,000 27,000 17,500
95,000 78,000 45,000 -
-
*Molecular weights (determined in 12% polyacrylamide gels)
based on earlier work (Morgan et al., 1973a; Whittaker et al., 1964; De Robertis et al., 1963), i.e., isolation of a nucleusfree and microsomefree crude mitochondrial fraction. In contrast to these workers no step was included to isolate synaptosomes, since synaptic vesicles have been shown to display extraordinarily low densities upon sucrose gradient centrifugation, permitting separation from heavier subcellular particles. Instead, the crude mitochondrial fraction was hypoosmotically shocked, thus releasing synaptic vesicles that were then separated from the bulk of other suborganelles by differential centrifugation in low-density sucrose. Finally, the crude vesicle fraction obtained was then substantially freed from large vesicular membrane fragments by passage through filters of decreasing pore size, a method that has been successfully applied by Morgan et al. (1973a) for total rat brain synaptic vesicles.
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The synaptic vesicle preparations of this paper were studied by electron microscopy and by enzyme assays. The results indicate that our fractions are about 70% pure, which is in agreement with the purity of synaptic vesicle fractions from guinea pig and rat brain as described by other workers(Morgan et al., 1973a;De Robertis et al., 1963; Soller et al., 1973). Electrophoretic analysis confirms that the synaptic vesicles have reduced complexity in their protein profile as contrasted with the membrane fraction of the total homogenate. Nevertheless, the protein profiles are rather complex even after reductive treatment, with 4 major protein bands each occupying 12-20% of total proteins. These high values suggest that the major proteins are intrinsic synaptic vesicle components. The origins of the minor protein components are difficult to assess, since they may be either of synaptic vesicular or of any other nature. The data of enzyme analyses point to contamination with synaptosomal plasma membranes because of elevated levels of (Na+-K+)ATPase. The results of our electrophoretic analyses are similar to the results of Morgan et al. (1973b). They found about 7 major proteins with molecular weight ranges similar to those of our major components. They, too, describe the existence of 3 glycoprotein bands, which had, however, molecular weights up to 120,000, in contrast to 95,000 in our preparation. On the one hand these differences may be due to abnormal behavior of glycoproteins upon SDS gel electrophotesis (Bretscher, 1973). On the other hand our own results (not published) indicate that synaptic vesicles prepared by our method from beef as well as from rat brain have membrane proteins that are very similar or are even identical. Thus, differences in protein patterns between the work of Morgan et al. (1973b) and our own work are probably related t o differences in preparation and degree of purity. The extraordinary heterogeneity of mammalian central nervous system (CNS) synaptic vesicles, which originates in both regional differences and in a sizable number of transmitters, has been a major difficulty in obtaining very pure preparations. This has become evident in our work as well as in others (Morgan et al., 1973a; Whittaker, 1973; Marchbanks, 1974). Initial anaiyses of synaptic vesicles from the electric organ of Torpedo (Whittaker et al., 1974) shows a different and somewhat simplified protein pattern as compared to mammalian CNS synaptic vesicles. However, this simplified protein pattern may possibly be due to separation conditions that are not optimal for fractionation of water-insoluble membrane proteins (Richter-Landsberg et al., 1974; Ruche1 et al., 1974). Nevertheless, the exclusively cholinergic nature of synaptic vesicles from Torpedo will in the long run be the most suitable material for detailed study of protein patterns in order to relate single proteins to functional roles in the exocytosis-endocytosis cycle during synaptic release of transmitters.
ACKNOWLEDGMENTS The authors wish to thank Mrs. A. Wolff for the preparation of electron microscopic photographs and Miss H. Fruh for excellent technical help. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 33). This work is part of the doctoral thesis of C. Richter-Landsberg, Gottingen, 1975.
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Whittaker, V.P., Dowdall, M.J., Dowe, G.H.C., Facino, R.M., and Scotto, J. (1974). Proteins of cholinergic synaptic vesicles from the electric organ of Torpedo : Characterization of a low molecular weight acidic protein. Brain Res. 75:115-131. Zacharius, R.M., Zell, T.E., Morrison, J.H., and Woodlock, J.J. (1969). Glycoprotein staining following electrophoresis on acrylamide gel. Anal. Biochem. 30:148-152. Zimmermann, H., and Whittaker, V.P. (1974a). Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapsis of the electric organ of Torpedo: A combined biochemical, electrophysiological and morphological study. J. Neurochem. 22 :435450. Zimmermann, H., and Whittaker, V.P. (1974b). Different recovery rates of the electrophysiological, biochemical and morphological parameters in the cholinergic synapses of the Torpedo electric organ after stimulation. J. Neurochem. 22:1109-1114.
