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The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: implications for vesicle structure
Brain Research, 161 (1979) 447-457 © Elsevier/North-Holland Biomedical Press
447
T H E LIPID A N D P R O T E I N C O N T E N T OF C H O L I N E R G I C SYNAPTIC VESICLES F R O M T H E ELECTRIC O R G A N OF Torpedo marmorata P U R I F I E D TO C O N S T A N T COMPOSITION: IMPLICATIONS FOR VESICLE STRUCTURE
KAZUAKI OHSAWA*, GORDON H. C. DOWE, STEPHEN J. MORRIS and VICTOR P. WHITTAKER** Abteilung Neurochemie, Max-Planck-Institutfiir Biophysikalische Chemic, Giittingen (G.F.R.) (Accepted June 1st, 1978)
SUMMARY The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 × 105 molecules/vesicle; the mean values, for all determinations, were 4.1 -t- S.E.M. 0.6 and 2.6 × 105 4- S.E.M. 0.6 × 105 respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2 % can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients3, 5.
INTRODUCTION Measurements 3,5 of the density, at different osmotic pressures and in density gradients formed from penetrating and non-penetrating solutes, of synaptic vesicles isolated from the cholinergic electromotor nerve terminals in the electric organ of Torpedo marmorata have led to conclusions about the structure of these vesicles which are not completely consistent with previously published valuesll, 21 for their lipid and * Department of Physiology,Faculty of Medicine, University of Tokyo, Bunkyo-Ku, Tokyo, Japan. ** To whom reprint requests should be addressed.
448 protein composition. Since the vesicle preparations previously obtained~l, ~6 were largely free from membrane fragments, as judged by electron microscopic examination, the main non-vesicular contaminant is likely to have been traces of adsorbed soluble protein, despite the fact that soluble cytoplasmic enzyme activities were reduced to negligibly low values 11. Accordingly, renewed attempts have been made to purify synaptic vesicles to maximum acetylcholine and ATP contents using constancy of composition with respect to these components as the criterion of purity. Accurate knowledge of the composition of synaptic vesicles is obviously essential for an understanding of the mechanism of storage and release of the transmitter. Some preliminary results have been presentedlL METHODS
Preparation of vesicles Vesicles were isolated by zonal centrifugation of cytoplasmic extracts of frozen, crushed electric tissue essentially as previously described7,11,21,24,26 with minor modifications, some of which, however, appear to be critical for the degree of purity of the vesicles obtained. Torpedones marrnoratae, kindly provided by the lnstitut de Biologic Marine, Arcachon, and kept in a sea-water aquarium at 17 °C, were anaesthetized by immersion in sea-water containing 0.05 ~ Tricaine (ethyl-m-aminobenzoate) methanesulphonate (MS 222, Sandoz); the electric organs were removed, frozen in liquid nitrogen and then crushed to a coarse powder. Vesicles, together with some soluble protein and small membrane fragments, were extracted from the powder while still frozen by suspending the latter in 0.4 M NaC1 or 0.8 M glycine containing 10 mM Tris.HCl buffer, pH 7.0 (100 ml/100 g of tissue); the suspension was squeezed through 4 layers of cheese cloth and the filtrate so obtained was centrifuged at 10,500 rev/min (104 × gave) for 30 min. After sampling, the supernatant (50 ml equivalent to about 70 g of tissue) was layered on a sucrose density gradient in a zonal rotor (Beckman Ti 14) of the same form and under the same conditions as previously21, except that the sample was overlaid with extractant (50 ml), the initial step of the gradient was prolonged to 400 ml (cf. ref. 24), 10 mM Tris.HCl buffer, pH 7.0, was added to all solutions and the osmolarity of sucrose solutions below 0.8 M was made up to approximately 0.8 Osm by the addition of either NaC1 or glycine (in twice the concentration of NaCI) 25 as indicated. The vesicles were identified in the gradient by their acetylcholine and ATP content7,el; acetylcholinesterase was used as a marker for both soluble protein and membrane fragments21. Extraction of the crushed organ while still frozen instead of after thawing to 0 °C2~ made little difference to the yield of acetylcholine but greatly reduced the amount of soluble protein extracted, giving vesicle peaks with a much higher acetylcholine content/mg of protein than obtained earlier. By contrast, the substitution of NaC1 by glycine made little difference to the purity of the vesicles in the initial separation, but appeared to be essential when the vesicles were repurified in a second zonal separation. Recoveries of acetylcholine and ATP in each purification step were 80 ~ or better.
449
Purification of vesicles In some experiments vesicles separated in an initial zonal run were submitted to a second zonal separation using a sucrose-glycine gradient. Usually the peak fraction and the two fractions on each side (all of 10 ml) were pooled after sampling (2 ml/fraction), diluted with iso-osmotic NaC1 or glycine so as to bring the concentration of sucrose to 0.2 M, and then placed on the second gradient. The new vesicle peak was again identified by its acetylcholine and ATP content. In other experiments the pooled vesicle peak (15 ml) was submitted to chromatography on columns (1.2 × 100 cm) of porous glass beads (CPG-10-3000, 120-200 mesh, pore diameter 3125 -q- S.D. 15 nm, Electro-Nucleonics, Clifton, N.J., treated before use with 1% Carbowax 20 M) essentially as previously described u, except the eluant was 0.8 M glycine containing 10 m M Tris.HCl, pH 7.0. The columns were calibrated after vesicle separations with polystyrene beads of diameter 315 and 90 nm; the latter had a retention volume close to that of the vesicles and appreciably higher than that of the larger beads. To maintain flow rates, columns were well washed and repacked between separations, especially if polystyrene beads had been used. The vesicle peak was again identified by its acetylcholine and ATP content. Recoveries were 80 % or more for both purification steps.
Analytical methods ATP was measured by the luciferin-luciferase method (ref. 7 modified from ref. 17), acetylcholine on a small slip of the dorsal muscle of the leech 20, acetylcholinesterase by a colorimetric method 8, phospholipid as previously described 11 and protein by the Amido Schwarz method 14, using crystalline bovine serum albumin (Serva, Heidelberg) as standard. It was found necessary to use at least 1 ml of zonal vesicle fraction (10 ml) for a reliable protein determination. Fractions (1 ml) containing vesicles were treated with an equal volume of glutaraldehyde (5 ~o in 0.4 M cacodylate buffer, pH 7.0) and the fixed vesicles collected on a filter (Millipore VMWP 02500, 19 mm in effective diameter and 50 nm in mean pore size) by gentle suction. The filter holder was levelled before use by means of a spirit-level to ensure an even deposition and a uniform thickness ( _ 2 #m) of the vesicle pellicle. The upper surface of the pellicle was protected by covering it with a 1 mm thick layer of 4 ~ w/v agar, and the filter, with its layer of agar-covered vesicles, was transferred to a flat dish for osmication, dehydration and embedding. For electron microscopy, grey sections (about 100 nm thick) were cut vertically to the plane of the filter and viewed after lead staining. Such a section is shown in Fig. la. Evaluation of vesicle profiles. The distribution of equivalent circular profile diameters of the vesicle profiles seen in such sections was determined by means of a Zeiss Particle Size Analyser and the number of vesicles in the section was estimated in two ways. The first used a modification of a procedure described by Baudhuin and BerthetL As expected, the distribution of the profile diameters (Fig. 1b) was markedly skewed due to the presence in the field of non-equatorial vesicle sections. Because of the thickness of the section relative to the mean diameters of the vesicles and the greater contrast generated by regions of membrane whose surfaces lie parallel to the
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Fig. 1. Evaluation of vesicle profiles, a: cross-section of vesicle pellet obtained by filtration at (1) low magnification showing uniformity of thickness and (2) at higher magnification showing vesicle profiles. b: distribution of vesicle profiles. The heavy line is the observed distribution of 618 profile diameters in an electron micrograph a portion of which is shown in a. Distributions I-V are the theoretical distributions of profile diameters corresponding to blocks 1-5 on the assumption that half of the profiles that are not accounted for by sections of larger profiles are lyingwith their centres in the section, and therefore project as median (equatorial) sections. Note that in the range of the smaller profile diameters (blocks 6-10) some profiles have not been observed (vertically hatched area) due to the well-known difficulty of detecting grazing (polar) sections; however, the missing profiles only amount to 7.6 % of the observed distribution, c: distribution of the 366 profile diameters remainingafter deducting profiles representing sections of vesicles whose centres lie outside the section, d: a plot of frequency versus diameters using a linear scale of probits shows that these diameters are approximately normal in distribution.
electron beam, vesicles that lie wholly or mainly within the section project as m e d i a n sections; thus there are more such sections in the d i s t r i b u t i o n t h a n would theoretically be predicted if the thickness of the section were small c o m p a r e d to the radius of the vesicles as in the case considered by B a u d h u i n a n d Berthet z. The chance of the centre of a vesicle lying within the section is t/(t+D) where D is the diameter of the vesicle a n d t the section thickness; where t _~ D, as here, t/(t+ D) ~_ 0.5. Accordingly half the profiles of greatest diameter (Fig. l b l ) were assumed to arise from such vesicles a n d the other half to represent m e d i a n sections. The Wicksell distribution23 was derived from the latter in order to estimate the c o n t r i b u t i o n made
451 by non-median sections of vesicles of this diameter to profiles of smaller vesicles. After the contribution of such sections to the next smaller size range of profiles (Fig. 1b2) had been deducted, the correction process was reiterated. In all, 5 Wicksell corrections were applied, as shown in Fig. lb, thus enabling the number n and size distribution of the vesicle population whose centres lie within the section to be determined (Fig. Ic). From the latter the mean diameter of the vesicle population and its variance was obtained; it will be seen that, unlike the profile diameters, those of the vesicles are symmetrical and approximately normal (Fig. ld) in distribution with a relatively small dispersion. The total number of vesicles in 1 ml was then calculated as Ahn/at where A is the area and h the thickness of the pellicle derived from 1 ml of vesicle suspension, a is the area of the field studied, t is the section thickness and n, as before, is the number of vesicles whose centres lie within the volume of the section corresponding to a. The number of such vesicles was 52 q- S.E.M. 5 ~o of the total in the 5 preparations studied. No attempt was made to calibrate the magnification of the electron microscope or to correct for swelling or shrinking during preparation for electron microscopy; thus the absolute values for profile diameters are not likely to be accurate to more than 10%. RESULTS Table I gives the acetylcholine, ATP and lipid content of, and the number of vesicles present in, the various vesicle fractions per mg of protein; the results are compared with earlier work in Table II. It will be seen that the purest preparations now obtainable have an acetyleholine content, relative to protein, about 10 times higher than our first zonal fractions 21, and over 100 times greater than our first step gradient fractions 15. By contrast, the preparation 9 oddly described in a recent paper t0 as the first pure vesicle fraction was, in fact, little different, in either acetylcholine content or morphology, from the one t5 on which, with minor modifications, it was based. As far as the present work is concerned, little increase in vesicle numbers or additional purification of other vesicle constituents relative to protein was obtained by rerunning vesicles separated in a zonal rotor on a second gradient or filtering them through columns of porous glass beads. For lipid the purification was never more than 1 9 ~ and - - taking the results as a whole - - was not significant. For the more diffusible acetylcholine a significant (P < 0.02) loss occurred as a result of a second zonal run. These findings are in contrast to earlier work it, and appear to result from the superior resolving power of the modified initial stages of the preparative procedure. Thus the vesicles can be regarded as essentially pure after the first zonal separation by the usual criterion of purification to constant specific content and any non-vesicular protein if still present must be adsorbed very tenaciously. The lipid: protein ratio is higher than previously observed ~1, again suggesting that the vesicles have been freed from extraneous protein present in the earlier preparations. In more recent work from this laboratory 19, somewhat higher values for the
4*** 3 2§ 9
Zi Z2 G All
1.61 ~ 0.03 1.77 ± 0.05 1.71 (t) 1.68 ± 0.04 (8)
Lipid** (g)
4.45 2.21 6.10 4.07
± ~ ± !
0.50§§ 0.41§§ 0.90 0.59
0.62 0.34 0.71 0.55
± ± ~ m
0.13 0.06§§ 0.0l§§ 0.07
Concentration* (units/g o f protein) Acetylcholine ATP (rnmole) (mmole) 0.86 ± 0.12 (2) 1.60 (1) 1.44 ± 0.31 1.24 ± 0.12 (5)
33.9 10.1 26.0 26.0
± 11.8 (2) (1) t : 1.5 i 5.8 (5)
90.7 86.2 86.4 88.0
± 0.5 (2) (1) ± 0.3 ± 1.1 (5)
No o f acetylDiameter (nm) Vesicles (number choline molecules/ x 10-16) vesicle ( × 10-a)
503 94 145 --
Total no. of profiles measured
* Values refer to fraction with highest ATP content/ml. ** Phospholipid + cholesterol; phospholipid values were multiplied by 1.22, a factor based on a mol.wt, for cholesterol of 385, a mean mol.wt, for phospholipid of 750 and a cholesterol: phospholipid molar ratio of 0.4211. *** ZI utilized sucrose-sodium chloride gradient; all other zonal runs were with sucrose-glycine, ZI in these experiments used sucrose-sodium chloride gradients. §§ Means significantly different ( P < 0.02) by Walsh's and Lord's tests 6
No. of exps.
Type of separation
Figures are single values or means f range (2) or S.E.M. (3 or more experiments; numbers are given in parentheses when different from those in col. 2). Abbreviations: Z1, first zonal run; Z2, second zonal run; G, glass-bead chromatography.
Lipid, acetylcholine and ATP contents o f cholinergic synaptic vesicles from Torpedo
TABLE I
4~ L~ to
SG SG Z
Z Z Z + G All results Z + G SG + Z
15 9
26 11
VP ZVP-T GVP-T (vesicle peak) (vesicle peak) (vesicle peak)
II 2 VP
Fraction designation
-0.69 ± 0.03 (3) -1.68 ± 0.04 (8) 1.71 3.47
----
Lipid (g)
1.11 (6)§§ 0.08 (4)§§§ 0.11 (4)§§§ 0.59 (9) 0.90 (2) 0.63 (6)
--1.24 ~ 0.12 (5) 1.44 ± 0.31 (2) --
0.52§
0.57 :i: 0.07 (4) ± ± ± ± ± ±
--
2.31 0.29 0.79 4.07 6.10 6.90
--
0.17"**
× 10 -16)
Vesicles (number
0.05*
Acetylcholine (mmole)
Concentration (units/g of protein)
26.0 ~ 5.8 (5) 26.0 i 1.5 (2)
6.6 ± 3.0 (4) 13.7 (max)
4.0
88.0 4- 1.1 (5) 86.4 :k 0.3 (2)
--
83 ± 28**
No. of acetylDiameter (nm) choline molecules ( x lO-4)/vesicle
Calculated from data ofref. 15, Table 3. Mean ± S.D. for a population of 115 vesicles. Calculated from data o f ref. 9, Table 2. Calculated from mean no. of acetylcholine molecules/vesicle (ref. 21, Table 5) and mean acetylcholine content/m§ of protein corrected for nonvesicular protein (ref. 21, Table 4). §§ Calculated from ref. 26, Table 3; see note at foot of p. 305, ref. 11. §§§ Results low due to use of stored tissue.
* ** *** §
18
This paper
21
Type of preparation
Reference
Values are single determinations or means ± range (2) or S.E.M. (3 or more experiments; nos. in parentheses). Abbreviations: SG, step gradient; Z, gradient separation in zonal rotor; G, chromatography on porous glass beads.
Comparison of results with previous work
TABLE II
td~
454 acetylcholine and lipid content/g of protein have been obtained (Table 1I, last line) in preparations in which the vesicles were extracted from the frozen tissue and concentrated on a step gradient in the presence of 3.5 m M EGTA and 0.3 m M phenylmethylsulphonylfluoride, a protease inhibitor, before separation in a zonal rotor. This suggests that our vesicle preparations still contained residual amounts of adsorbed surface protein which are removed in the presence of EGTA and that the acetylcholine/lipid ratio, which is 3.55 (zonal followed by glass-bead chromatography) or 2.42 (all results) mmole/g for our preparations and 2.00 for the EGTA preparations, may be a more reliable criterion of vesicle integrity. It remains to be determined whether the EGTA-removable protein is intrinsic vesicle protein or adsorbed cytoplasmic protein and, if the lattel, whether specific protein(s) (e.g. filamentous proteins) are involved or not. DISCUSSION
The average amounts of acetylcholine, ATP, lipid and protein present per vesicle can be readily calculated from the results of Table I and are presented in Table III. We may now calculate the volume that would be occupied by the observed amount of lipid utilizing Tanford's is values for the area occupied by the polar head groups of phospholipid and cholesterol. For 135 ag of phospholipid and cholesterol in a molar ratio 1~ of 0.42 this is 7.0 × 104 nm z. Arranged as a bilayer of thickness 4.3 nm is, it could exactly envelop a core of diameter 101.2 nm to give a sphere of 110 nm in diameter. This is about 25 ~ greater than the measured values for fixed, dehydrated and embedded vesicles (Table I, col. 8). Such a pure lipid membrane would occupy 22 ~o v/v of the vesicle and would have a density of 135 × 10 -18 g/vol, of membrane in ml or 0.90 g/ml. This is less than the value of 1.016 measured by Newman and Huang lz for bilayers of lecithin and cholesterol in the ratio t :0.4. The latter value implies a smaller mean head group area than that given by Tanford, and thus a smaller calculated vesicle diameter (96.5 nm, within 10 ~ of the observed value) and a larger membrane volume fraction (0.244). The inclusion of about 35 ~ w/w (72 ag/vesicle) of protein of assumed density 1.35 g/ml in the memblane would bring its density to the observed value of 1.1325, leaving 9 ag/vesicle for the core. How the protein is inserted into the membrane makes relatively little difference to the diameter. Thus, if 50 ~,, is surface protein and 50 ~,, is inserted as integral membrane protein in the form of TABLE III
Amount of vesicle components per vesicle Values are derived from the means of all determinations in Table I.
Component
Unit
Amount/vesicle
Acetylcholine ATP Lipid Protein
molecules ( × 10 -4) molecules ( × 10-4) ag ag
19.9 2.7 13 5 81
455 TABLE IV Calculated and observed densities o f vesicles under different conditions Condition
Membrane Intact vesicle Vesicle 'ghost' Membrane + non-diffusiblecore
Density (glml) Calculated from data in this paper
Observed values from re]: 5
1.132* 1.052 1.034 I. 142
1.132 1.058 1.035 1.168
* Set equal to the observed value (col. 3). TABLE V Comparisons of parameters calculated from analytical and density gradient results
The two columns under Ref. 5 refer to calculations based on two different sets of assumptions. Volume fraction defined as the volume of a specificcompartment expressedas a fraction of total vesiclevolume. Parameter
This paper Ref 5
Volume fraction of membrane
0.231i
core protein 0.0121 core water 0.757 Radius of vesicle(rim) at 51 (4.3) assumed lipid bilayer thick- -ness (nm) given in parentheses
i0.262 0.21 0.79 53 (4.0) --
i0.084 0.654 50 (4.8) 40 (3.9)
cylinders 7 nm in length, it would only increase the effective vesicle diameter by 6.1 nm and decrease the membrane volume fraction by 5.3 ~ (to 0.231). At an assumed partial specific volume of 0.74, the core protein would occupy 1.53 ~ of the core or 1.18 ~ of the vesicle, leaving 75.7 ~ to be filled with water and low molecular weight solutes. If the 199,000 molecules of acetylcholine and 27,000 molecules of ATP (Table III) are dissolved in this space, their combined molar concentration would be 1.0 M, slightly hyperosmotic to Torpedo body fluids (830 Osm). The core protein (or proteoglycan16), even if its molecular weight is only 10,00016,22, would contribute little more to this (about 0.01 M). Assuming a density for the liquid core based on the acetylcholine and ATP content 5 of 1,024, we may calculate the density of the hydrated vesicle as 0.231 × 1.132 ÷ 0.012 × 1.35 + 0.757 x 1.024 = 1.052, which is rather close to the observed value (1.0585). Similarly, we may calculate the density of a vesicle ghost, in which the aqueous core containing diffusible solutes is assumed to be replaced by water without alteration of vesicle dimensions, and that of the membrane plus core protein, which should equal the density of water-shocked vesicles in a penetrating gradient. The results are summarized in Tables IV and V, from which it will be seen that the values calculated from our analyses are reasonably consistent with our recent density gradient studies 3,5. The main uncertainty in the calculations is the correct value to be assigned to the densities
456 o f lipid and protein; if the values previously assumed (lipid, 1.11; protein, 1.33) are used, densities for m e m b r a n e + core, ghosts a n d intact vesicles are 2 ~ o higher. Nevertheless, the results clearly s u p p o r t the conclusion drawn from the density gradient studies, n a m e l y that the cholinergic synaptic vesicle from T o r p e d o electric organs is a highly h y d r a t e d structure storing acetylcholine and A T P in osmotically active form a n d with a relatively small content o f m a c r o m o l e c u l a r core material. The mechanism whereby acetylcholine and A T P are a c c u m u l a t e d in such high concentration within the s e m i p e r m e a b l e lipoprotein vesicle m e m b r a n e is n o t yet known, t h o u g h o t h e r recent results f r o m this l a b o r a t o r y 4 suggest that a t r a n s p o r t A T P a s e m a y be involved, as in the chromaffin granule 1. W h e t h e r these findings a p p l y equally to m a m m a l i a n cholinergic vesicles, which are c o n s i d e r a b l y smaller and a p p e a r to store much less acetylcholine H, will require further investigation. ACKNOWLEDGEMENTS K. O h s a w a gratefully a c k n o w l e d g e s the award, in 1975, o f a p o s t d o c t o r a l s t i p e n d i u m by the Max-Planck-Gesellschaft. W e wish to t h a n k Dr. D e r e k M a r s h for helpful discussions. REFERENCES l Bashford, C. L., Casey, R. P., Radda, G. K. and Richie, G. A., Energy-coupling in adrenal chromaffin granules, Neuroscience, 1 (1976)399~,12. 2 Baudhuin, P. and Berthet, J., Electron microscopic examination of fractions. II. Quantitative analysis of the mitochondrial population isolated from rat liver, J. Cell Biol., 35 (1967) 631-648. 3 Breer, H. and Morris, S. J., A structural model of the Torpedo electric organ synaptic vesicles from buoyant density measurements, Abstr. int. Meet. Soc. Neurochem., Copenhagen, 1977, p. 200. 4 Breer, H., Morris, S. J. and Whittaker, V. P., Adenosine triphosphatase activity associated with purified cholinergic synaptic vesicles of Torpedo marmorata, Earop. J. Biochem., 80 (1977) 313-3 ! 8. 5 Breer, H., Morris, S. J. and Whittaker, V. P., The structure of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures, Europ. J. Biochem., 80 (1977) 313-318. 6 Diem, K. (Ed.), Documenta Geigy: Scientific Tables, 6th Ed., Geigy Pharmaceuticals, Ardsley, New York, 1962, p. 53. 7 Dowdall, M. J., Boyne, A. F. and Whittaker, V. P., Adenosine triphosphate a constituent of cholinergic synaptic vesicles, Biochem. J., 140 (1974) 1-12. 8 Ellman, G. L., Courtney, D. K., Andres, V. and Featherstone, R. M., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. PharmacoL, 7 (1961) 88-95. 9 Israel, M., Gautron, J. et Lesbats, B., Fractionnement de l'organe dlectrique de la torpille: localisa tion subcellulaire de rac6tylcholine, J. Neurochem., 17 (1970) 1441-1450. l0 Morel, N., Israel, M., Manaranche, R. and Mastour-Franchon, P., Isolation of pure cholinergic nerve endings from Torpedo electric organ, J. Cell Biol., 75 (1977) 43-55. I l Nagy, A., Baker, R. R., Morris, S. J. and Whittaker, V. P., The preparation and characterization of synaptic vesicles of high purity, Brain Research, 109 (1976) 285-309. 12 Newman, G. C. and Huang, C., Structural studies on phosphatidylcholine-cholesterolmixed vesicles, Biochemistry, 14 (1975) 3363-3370. 13 Ohsawa, K., Dowe, G. H. C., Morris, S. J. and Whittaker, V. P., Preparation of ultra-pure synaptic vesicles from the electric organ of Torpedo marmorata by porous glass bead chromatography and estimation of their acetylcholine content, Exp. Brain Res., 24 (1976) 19. 14 Scha.ffner, W. and Weissmann, C., A rapid, sensitive and specific method for the determination of protein in dilute solution, Analyt. Biochem., 56 (1973) 502-514. 15 Sheridan, M. N., Whittaker, V. P. and Israel, M., The subcellular fractionation of the electric organ of Torpedo, Z. Zellforsch., 74 (1966) 281-307.
457 16 Stadler, H. and Whittaker, V. P., Identification of vesiculin as a glycoaminoglycan, Brain Research (1979) in press. 17 Stanley, P. E. and Williams, S. G., Use of liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme, Analyt. Biochem., 29 (1969) 381-392. 18 Tanford, C., The Hydrophobic Effect: Formation of Micelles and Biological Membranes, J. Wiley and Sons, New York, 1973. 19 Tashiro, T. and Stadler, H., Proteins of cholinergic synaptic vesicles of Torpedo marmorata, Europ. J. Biochem., (1979) in press. 20 Whittaker, V. P. and Barker, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, Methods Neurochem., 2 (1972) 1-52. 21 Whittaker, V. P., Essman, W. B. and Dowe, G. H. C., The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae, Biochem. J., 128 (1972) 833-846. 22 Whittaker, V. P., Dowdall, M. J., Dowe, G. H. C., Facino, R. M. and Scotto, J., Proteins of cholinergic synaptic vesicles from the electric organ of Torpedo: characterization of a low molecular weight acidic protein, Brain Research, 75 (1974) 115-131. 23 Wicksell, S. D., The corpuscle problem. A mathematical study o f a biometric problem, Biometrika, 17 (1925) 84-99. 24 Zimmermann, H. and Denston, C. R., Separation of vesicles of different functional states from the cholinergic synapses of the Torpedo electric organ, Neuroscience, 2 (1977) 715-730. 25 Zimmermann, A. and Dowdall, M. J., Vesicular storage and release of a false cholinergic transmitter (acetylpyrrolcholine) in the Torpedo electric organ, Neuroscience, 2 (1977) 731-739. 26 Zimmermann, H. and Whittaker, V. P., Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo: a combined biochemical, electrophysiological and morphological study, J. Neurochem., 22 (1974) 435-450.
447
T H E LIPID A N D P R O T E I N C O N T E N T OF C H O L I N E R G I C SYNAPTIC VESICLES F R O M T H E ELECTRIC O R G A N OF Torpedo marmorata P U R I F I E D TO C O N S T A N T COMPOSITION: IMPLICATIONS FOR VESICLE STRUCTURE
KAZUAKI OHSAWA*, GORDON H. C. DOWE, STEPHEN J. MORRIS and VICTOR P. WHITTAKER** Abteilung Neurochemie, Max-Planck-Institutfiir Biophysikalische Chemic, Giittingen (G.F.R.) (Accepted June 1st, 1978)
SUMMARY The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 × 105 molecules/vesicle; the mean values, for all determinations, were 4.1 -t- S.E.M. 0.6 and 2.6 × 105 4- S.E.M. 0.6 × 105 respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2 % can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients3, 5.
INTRODUCTION Measurements 3,5 of the density, at different osmotic pressures and in density gradients formed from penetrating and non-penetrating solutes, of synaptic vesicles isolated from the cholinergic electromotor nerve terminals in the electric organ of Torpedo marmorata have led to conclusions about the structure of these vesicles which are not completely consistent with previously published valuesll, 21 for their lipid and * Department of Physiology,Faculty of Medicine, University of Tokyo, Bunkyo-Ku, Tokyo, Japan. ** To whom reprint requests should be addressed.
448 protein composition. Since the vesicle preparations previously obtained~l, ~6 were largely free from membrane fragments, as judged by electron microscopic examination, the main non-vesicular contaminant is likely to have been traces of adsorbed soluble protein, despite the fact that soluble cytoplasmic enzyme activities were reduced to negligibly low values 11. Accordingly, renewed attempts have been made to purify synaptic vesicles to maximum acetylcholine and ATP contents using constancy of composition with respect to these components as the criterion of purity. Accurate knowledge of the composition of synaptic vesicles is obviously essential for an understanding of the mechanism of storage and release of the transmitter. Some preliminary results have been presentedlL METHODS
Preparation of vesicles Vesicles were isolated by zonal centrifugation of cytoplasmic extracts of frozen, crushed electric tissue essentially as previously described7,11,21,24,26 with minor modifications, some of which, however, appear to be critical for the degree of purity of the vesicles obtained. Torpedones marrnoratae, kindly provided by the lnstitut de Biologic Marine, Arcachon, and kept in a sea-water aquarium at 17 °C, were anaesthetized by immersion in sea-water containing 0.05 ~ Tricaine (ethyl-m-aminobenzoate) methanesulphonate (MS 222, Sandoz); the electric organs were removed, frozen in liquid nitrogen and then crushed to a coarse powder. Vesicles, together with some soluble protein and small membrane fragments, were extracted from the powder while still frozen by suspending the latter in 0.4 M NaC1 or 0.8 M glycine containing 10 mM Tris.HCl buffer, pH 7.0 (100 ml/100 g of tissue); the suspension was squeezed through 4 layers of cheese cloth and the filtrate so obtained was centrifuged at 10,500 rev/min (104 × gave) for 30 min. After sampling, the supernatant (50 ml equivalent to about 70 g of tissue) was layered on a sucrose density gradient in a zonal rotor (Beckman Ti 14) of the same form and under the same conditions as previously21, except that the sample was overlaid with extractant (50 ml), the initial step of the gradient was prolonged to 400 ml (cf. ref. 24), 10 mM Tris.HCl buffer, pH 7.0, was added to all solutions and the osmolarity of sucrose solutions below 0.8 M was made up to approximately 0.8 Osm by the addition of either NaC1 or glycine (in twice the concentration of NaCI) 25 as indicated. The vesicles were identified in the gradient by their acetylcholine and ATP content7,el; acetylcholinesterase was used as a marker for both soluble protein and membrane fragments21. Extraction of the crushed organ while still frozen instead of after thawing to 0 °C2~ made little difference to the yield of acetylcholine but greatly reduced the amount of soluble protein extracted, giving vesicle peaks with a much higher acetylcholine content/mg of protein than obtained earlier. By contrast, the substitution of NaC1 by glycine made little difference to the purity of the vesicles in the initial separation, but appeared to be essential when the vesicles were repurified in a second zonal separation. Recoveries of acetylcholine and ATP in each purification step were 80 ~ or better.
449
Purification of vesicles In some experiments vesicles separated in an initial zonal run were submitted to a second zonal separation using a sucrose-glycine gradient. Usually the peak fraction and the two fractions on each side (all of 10 ml) were pooled after sampling (2 ml/fraction), diluted with iso-osmotic NaC1 or glycine so as to bring the concentration of sucrose to 0.2 M, and then placed on the second gradient. The new vesicle peak was again identified by its acetylcholine and ATP content. In other experiments the pooled vesicle peak (15 ml) was submitted to chromatography on columns (1.2 × 100 cm) of porous glass beads (CPG-10-3000, 120-200 mesh, pore diameter 3125 -q- S.D. 15 nm, Electro-Nucleonics, Clifton, N.J., treated before use with 1% Carbowax 20 M) essentially as previously described u, except the eluant was 0.8 M glycine containing 10 m M Tris.HCl, pH 7.0. The columns were calibrated after vesicle separations with polystyrene beads of diameter 315 and 90 nm; the latter had a retention volume close to that of the vesicles and appreciably higher than that of the larger beads. To maintain flow rates, columns were well washed and repacked between separations, especially if polystyrene beads had been used. The vesicle peak was again identified by its acetylcholine and ATP content. Recoveries were 80 % or more for both purification steps.
Analytical methods ATP was measured by the luciferin-luciferase method (ref. 7 modified from ref. 17), acetylcholine on a small slip of the dorsal muscle of the leech 20, acetylcholinesterase by a colorimetric method 8, phospholipid as previously described 11 and protein by the Amido Schwarz method 14, using crystalline bovine serum albumin (Serva, Heidelberg) as standard. It was found necessary to use at least 1 ml of zonal vesicle fraction (10 ml) for a reliable protein determination. Fractions (1 ml) containing vesicles were treated with an equal volume of glutaraldehyde (5 ~o in 0.4 M cacodylate buffer, pH 7.0) and the fixed vesicles collected on a filter (Millipore VMWP 02500, 19 mm in effective diameter and 50 nm in mean pore size) by gentle suction. The filter holder was levelled before use by means of a spirit-level to ensure an even deposition and a uniform thickness ( _ 2 #m) of the vesicle pellicle. The upper surface of the pellicle was protected by covering it with a 1 mm thick layer of 4 ~ w/v agar, and the filter, with its layer of agar-covered vesicles, was transferred to a flat dish for osmication, dehydration and embedding. For electron microscopy, grey sections (about 100 nm thick) were cut vertically to the plane of the filter and viewed after lead staining. Such a section is shown in Fig. la. Evaluation of vesicle profiles. The distribution of equivalent circular profile diameters of the vesicle profiles seen in such sections was determined by means of a Zeiss Particle Size Analyser and the number of vesicles in the section was estimated in two ways. The first used a modification of a procedure described by Baudhuin and BerthetL As expected, the distribution of the profile diameters (Fig. 1b) was markedly skewed due to the presence in the field of non-equatorial vesicle sections. Because of the thickness of the section relative to the mean diameters of the vesicles and the greater contrast generated by regions of membrane whose surfaces lie parallel to the
450
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Fig. 1. Evaluation of vesicle profiles, a: cross-section of vesicle pellet obtained by filtration at (1) low magnification showing uniformity of thickness and (2) at higher magnification showing vesicle profiles. b: distribution of vesicle profiles. The heavy line is the observed distribution of 618 profile diameters in an electron micrograph a portion of which is shown in a. Distributions I-V are the theoretical distributions of profile diameters corresponding to blocks 1-5 on the assumption that half of the profiles that are not accounted for by sections of larger profiles are lyingwith their centres in the section, and therefore project as median (equatorial) sections. Note that in the range of the smaller profile diameters (blocks 6-10) some profiles have not been observed (vertically hatched area) due to the well-known difficulty of detecting grazing (polar) sections; however, the missing profiles only amount to 7.6 % of the observed distribution, c: distribution of the 366 profile diameters remainingafter deducting profiles representing sections of vesicles whose centres lie outside the section, d: a plot of frequency versus diameters using a linear scale of probits shows that these diameters are approximately normal in distribution.
electron beam, vesicles that lie wholly or mainly within the section project as m e d i a n sections; thus there are more such sections in the d i s t r i b u t i o n t h a n would theoretically be predicted if the thickness of the section were small c o m p a r e d to the radius of the vesicles as in the case considered by B a u d h u i n a n d Berthet z. The chance of the centre of a vesicle lying within the section is t/(t+D) where D is the diameter of the vesicle a n d t the section thickness; where t _~ D, as here, t/(t+ D) ~_ 0.5. Accordingly half the profiles of greatest diameter (Fig. l b l ) were assumed to arise from such vesicles a n d the other half to represent m e d i a n sections. The Wicksell distribution23 was derived from the latter in order to estimate the c o n t r i b u t i o n made
451 by non-median sections of vesicles of this diameter to profiles of smaller vesicles. After the contribution of such sections to the next smaller size range of profiles (Fig. 1b2) had been deducted, the correction process was reiterated. In all, 5 Wicksell corrections were applied, as shown in Fig. lb, thus enabling the number n and size distribution of the vesicle population whose centres lie within the section to be determined (Fig. Ic). From the latter the mean diameter of the vesicle population and its variance was obtained; it will be seen that, unlike the profile diameters, those of the vesicles are symmetrical and approximately normal (Fig. ld) in distribution with a relatively small dispersion. The total number of vesicles in 1 ml was then calculated as Ahn/at where A is the area and h the thickness of the pellicle derived from 1 ml of vesicle suspension, a is the area of the field studied, t is the section thickness and n, as before, is the number of vesicles whose centres lie within the volume of the section corresponding to a. The number of such vesicles was 52 q- S.E.M. 5 ~o of the total in the 5 preparations studied. No attempt was made to calibrate the magnification of the electron microscope or to correct for swelling or shrinking during preparation for electron microscopy; thus the absolute values for profile diameters are not likely to be accurate to more than 10%. RESULTS Table I gives the acetylcholine, ATP and lipid content of, and the number of vesicles present in, the various vesicle fractions per mg of protein; the results are compared with earlier work in Table II. It will be seen that the purest preparations now obtainable have an acetyleholine content, relative to protein, about 10 times higher than our first zonal fractions 21, and over 100 times greater than our first step gradient fractions 15. By contrast, the preparation 9 oddly described in a recent paper t0 as the first pure vesicle fraction was, in fact, little different, in either acetylcholine content or morphology, from the one t5 on which, with minor modifications, it was based. As far as the present work is concerned, little increase in vesicle numbers or additional purification of other vesicle constituents relative to protein was obtained by rerunning vesicles separated in a zonal rotor on a second gradient or filtering them through columns of porous glass beads. For lipid the purification was never more than 1 9 ~ and - - taking the results as a whole - - was not significant. For the more diffusible acetylcholine a significant (P < 0.02) loss occurred as a result of a second zonal run. These findings are in contrast to earlier work it, and appear to result from the superior resolving power of the modified initial stages of the preparative procedure. Thus the vesicles can be regarded as essentially pure after the first zonal separation by the usual criterion of purification to constant specific content and any non-vesicular protein if still present must be adsorbed very tenaciously. The lipid: protein ratio is higher than previously observed ~1, again suggesting that the vesicles have been freed from extraneous protein present in the earlier preparations. In more recent work from this laboratory 19, somewhat higher values for the
4*** 3 2§ 9
Zi Z2 G All
1.61 ~ 0.03 1.77 ± 0.05 1.71 (t) 1.68 ± 0.04 (8)
Lipid** (g)
4.45 2.21 6.10 4.07
± ~ ± !
0.50§§ 0.41§§ 0.90 0.59
0.62 0.34 0.71 0.55
± ± ~ m
0.13 0.06§§ 0.0l§§ 0.07
Concentration* (units/g o f protein) Acetylcholine ATP (rnmole) (mmole) 0.86 ± 0.12 (2) 1.60 (1) 1.44 ± 0.31 1.24 ± 0.12 (5)
33.9 10.1 26.0 26.0
± 11.8 (2) (1) t : 1.5 i 5.8 (5)
90.7 86.2 86.4 88.0
± 0.5 (2) (1) ± 0.3 ± 1.1 (5)
No o f acetylDiameter (nm) Vesicles (number choline molecules/ x 10-16) vesicle ( × 10-a)
503 94 145 --
Total no. of profiles measured
* Values refer to fraction with highest ATP content/ml. ** Phospholipid + cholesterol; phospholipid values were multiplied by 1.22, a factor based on a mol.wt, for cholesterol of 385, a mean mol.wt, for phospholipid of 750 and a cholesterol: phospholipid molar ratio of 0.4211. *** ZI utilized sucrose-sodium chloride gradient; all other zonal runs were with sucrose-glycine, ZI in these experiments used sucrose-sodium chloride gradients. §§ Means significantly different ( P < 0.02) by Walsh's and Lord's tests 6
No. of exps.
Type of separation
Figures are single values or means f range (2) or S.E.M. (3 or more experiments; numbers are given in parentheses when different from those in col. 2). Abbreviations: Z1, first zonal run; Z2, second zonal run; G, glass-bead chromatography.
Lipid, acetylcholine and ATP contents o f cholinergic synaptic vesicles from Torpedo
TABLE I
4~ L~ to
SG SG Z
Z Z Z + G All results Z + G SG + Z
15 9
26 11
VP ZVP-T GVP-T (vesicle peak) (vesicle peak) (vesicle peak)
II 2 VP
Fraction designation
-0.69 ± 0.03 (3) -1.68 ± 0.04 (8) 1.71 3.47
----
Lipid (g)
1.11 (6)§§ 0.08 (4)§§§ 0.11 (4)§§§ 0.59 (9) 0.90 (2) 0.63 (6)
--1.24 ~ 0.12 (5) 1.44 ± 0.31 (2) --
0.52§
0.57 :i: 0.07 (4) ± ± ± ± ± ±
--
2.31 0.29 0.79 4.07 6.10 6.90
--
0.17"**
× 10 -16)
Vesicles (number
0.05*
Acetylcholine (mmole)
Concentration (units/g of protein)
26.0 ~ 5.8 (5) 26.0 i 1.5 (2)
6.6 ± 3.0 (4) 13.7 (max)
4.0
88.0 4- 1.1 (5) 86.4 :k 0.3 (2)
--
83 ± 28**
No. of acetylDiameter (nm) choline molecules ( x lO-4)/vesicle
Calculated from data ofref. 15, Table 3. Mean ± S.D. for a population of 115 vesicles. Calculated from data o f ref. 9, Table 2. Calculated from mean no. of acetylcholine molecules/vesicle (ref. 21, Table 5) and mean acetylcholine content/m§ of protein corrected for nonvesicular protein (ref. 21, Table 4). §§ Calculated from ref. 26, Table 3; see note at foot of p. 305, ref. 11. §§§ Results low due to use of stored tissue.
* ** *** §
18
This paper
21
Type of preparation
Reference
Values are single determinations or means ± range (2) or S.E.M. (3 or more experiments; nos. in parentheses). Abbreviations: SG, step gradient; Z, gradient separation in zonal rotor; G, chromatography on porous glass beads.
Comparison of results with previous work
TABLE II
td~
454 acetylcholine and lipid content/g of protein have been obtained (Table 1I, last line) in preparations in which the vesicles were extracted from the frozen tissue and concentrated on a step gradient in the presence of 3.5 m M EGTA and 0.3 m M phenylmethylsulphonylfluoride, a protease inhibitor, before separation in a zonal rotor. This suggests that our vesicle preparations still contained residual amounts of adsorbed surface protein which are removed in the presence of EGTA and that the acetylcholine/lipid ratio, which is 3.55 (zonal followed by glass-bead chromatography) or 2.42 (all results) mmole/g for our preparations and 2.00 for the EGTA preparations, may be a more reliable criterion of vesicle integrity. It remains to be determined whether the EGTA-removable protein is intrinsic vesicle protein or adsorbed cytoplasmic protein and, if the lattel, whether specific protein(s) (e.g. filamentous proteins) are involved or not. DISCUSSION
The average amounts of acetylcholine, ATP, lipid and protein present per vesicle can be readily calculated from the results of Table I and are presented in Table III. We may now calculate the volume that would be occupied by the observed amount of lipid utilizing Tanford's is values for the area occupied by the polar head groups of phospholipid and cholesterol. For 135 ag of phospholipid and cholesterol in a molar ratio 1~ of 0.42 this is 7.0 × 104 nm z. Arranged as a bilayer of thickness 4.3 nm is, it could exactly envelop a core of diameter 101.2 nm to give a sphere of 110 nm in diameter. This is about 25 ~ greater than the measured values for fixed, dehydrated and embedded vesicles (Table I, col. 8). Such a pure lipid membrane would occupy 22 ~o v/v of the vesicle and would have a density of 135 × 10 -18 g/vol, of membrane in ml or 0.90 g/ml. This is less than the value of 1.016 measured by Newman and Huang lz for bilayers of lecithin and cholesterol in the ratio t :0.4. The latter value implies a smaller mean head group area than that given by Tanford, and thus a smaller calculated vesicle diameter (96.5 nm, within 10 ~ of the observed value) and a larger membrane volume fraction (0.244). The inclusion of about 35 ~ w/w (72 ag/vesicle) of protein of assumed density 1.35 g/ml in the memblane would bring its density to the observed value of 1.1325, leaving 9 ag/vesicle for the core. How the protein is inserted into the membrane makes relatively little difference to the diameter. Thus, if 50 ~,, is surface protein and 50 ~,, is inserted as integral membrane protein in the form of TABLE III
Amount of vesicle components per vesicle Values are derived from the means of all determinations in Table I.
Component
Unit
Amount/vesicle
Acetylcholine ATP Lipid Protein
molecules ( × 10 -4) molecules ( × 10-4) ag ag
19.9 2.7 13 5 81
455 TABLE IV Calculated and observed densities o f vesicles under different conditions Condition
Membrane Intact vesicle Vesicle 'ghost' Membrane + non-diffusiblecore
Density (glml) Calculated from data in this paper
Observed values from re]: 5
1.132* 1.052 1.034 I. 142
1.132 1.058 1.035 1.168
* Set equal to the observed value (col. 3). TABLE V Comparisons of parameters calculated from analytical and density gradient results
The two columns under Ref. 5 refer to calculations based on two different sets of assumptions. Volume fraction defined as the volume of a specificcompartment expressedas a fraction of total vesiclevolume. Parameter
This paper Ref 5
Volume fraction of membrane
0.231i
core protein 0.0121 core water 0.757 Radius of vesicle(rim) at 51 (4.3) assumed lipid bilayer thick- -ness (nm) given in parentheses
i0.262 0.21 0.79 53 (4.0) --
i0.084 0.654 50 (4.8) 40 (3.9)
cylinders 7 nm in length, it would only increase the effective vesicle diameter by 6.1 nm and decrease the membrane volume fraction by 5.3 ~ (to 0.231). At an assumed partial specific volume of 0.74, the core protein would occupy 1.53 ~ of the core or 1.18 ~ of the vesicle, leaving 75.7 ~ to be filled with water and low molecular weight solutes. If the 199,000 molecules of acetylcholine and 27,000 molecules of ATP (Table III) are dissolved in this space, their combined molar concentration would be 1.0 M, slightly hyperosmotic to Torpedo body fluids (830 Osm). The core protein (or proteoglycan16), even if its molecular weight is only 10,00016,22, would contribute little more to this (about 0.01 M). Assuming a density for the liquid core based on the acetylcholine and ATP content 5 of 1,024, we may calculate the density of the hydrated vesicle as 0.231 × 1.132 ÷ 0.012 × 1.35 + 0.757 x 1.024 = 1.052, which is rather close to the observed value (1.0585). Similarly, we may calculate the density of a vesicle ghost, in which the aqueous core containing diffusible solutes is assumed to be replaced by water without alteration of vesicle dimensions, and that of the membrane plus core protein, which should equal the density of water-shocked vesicles in a penetrating gradient. The results are summarized in Tables IV and V, from which it will be seen that the values calculated from our analyses are reasonably consistent with our recent density gradient studies 3,5. The main uncertainty in the calculations is the correct value to be assigned to the densities
456 o f lipid and protein; if the values previously assumed (lipid, 1.11; protein, 1.33) are used, densities for m e m b r a n e + core, ghosts a n d intact vesicles are 2 ~ o higher. Nevertheless, the results clearly s u p p o r t the conclusion drawn from the density gradient studies, n a m e l y that the cholinergic synaptic vesicle from T o r p e d o electric organs is a highly h y d r a t e d structure storing acetylcholine and A T P in osmotically active form a n d with a relatively small content o f m a c r o m o l e c u l a r core material. The mechanism whereby acetylcholine and A T P are a c c u m u l a t e d in such high concentration within the s e m i p e r m e a b l e lipoprotein vesicle m e m b r a n e is n o t yet known, t h o u g h o t h e r recent results f r o m this l a b o r a t o r y 4 suggest that a t r a n s p o r t A T P a s e m a y be involved, as in the chromaffin granule 1. W h e t h e r these findings a p p l y equally to m a m m a l i a n cholinergic vesicles, which are c o n s i d e r a b l y smaller and a p p e a r to store much less acetylcholine H, will require further investigation. ACKNOWLEDGEMENTS K. O h s a w a gratefully a c k n o w l e d g e s the award, in 1975, o f a p o s t d o c t o r a l s t i p e n d i u m by the Max-Planck-Gesellschaft. W e wish to t h a n k Dr. D e r e k M a r s h for helpful discussions. REFERENCES l Bashford, C. L., Casey, R. P., Radda, G. K. and Richie, G. A., Energy-coupling in adrenal chromaffin granules, Neuroscience, 1 (1976)399~,12. 2 Baudhuin, P. and Berthet, J., Electron microscopic examination of fractions. II. Quantitative analysis of the mitochondrial population isolated from rat liver, J. Cell Biol., 35 (1967) 631-648. 3 Breer, H. and Morris, S. J., A structural model of the Torpedo electric organ synaptic vesicles from buoyant density measurements, Abstr. int. Meet. Soc. Neurochem., Copenhagen, 1977, p. 200. 4 Breer, H., Morris, S. J. and Whittaker, V. P., Adenosine triphosphatase activity associated with purified cholinergic synaptic vesicles of Torpedo marmorata, Earop. J. Biochem., 80 (1977) 313-3 ! 8. 5 Breer, H., Morris, S. J. and Whittaker, V. P., The structure of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures, Europ. J. Biochem., 80 (1977) 313-318. 6 Diem, K. (Ed.), Documenta Geigy: Scientific Tables, 6th Ed., Geigy Pharmaceuticals, Ardsley, New York, 1962, p. 53. 7 Dowdall, M. J., Boyne, A. F. and Whittaker, V. P., Adenosine triphosphate a constituent of cholinergic synaptic vesicles, Biochem. J., 140 (1974) 1-12. 8 Ellman, G. L., Courtney, D. K., Andres, V. and Featherstone, R. M., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. PharmacoL, 7 (1961) 88-95. 9 Israel, M., Gautron, J. et Lesbats, B., Fractionnement de l'organe dlectrique de la torpille: localisa tion subcellulaire de rac6tylcholine, J. Neurochem., 17 (1970) 1441-1450. l0 Morel, N., Israel, M., Manaranche, R. and Mastour-Franchon, P., Isolation of pure cholinergic nerve endings from Torpedo electric organ, J. Cell Biol., 75 (1977) 43-55. I l Nagy, A., Baker, R. R., Morris, S. J. and Whittaker, V. P., The preparation and characterization of synaptic vesicles of high purity, Brain Research, 109 (1976) 285-309. 12 Newman, G. C. and Huang, C., Structural studies on phosphatidylcholine-cholesterolmixed vesicles, Biochemistry, 14 (1975) 3363-3370. 13 Ohsawa, K., Dowe, G. H. C., Morris, S. J. and Whittaker, V. P., Preparation of ultra-pure synaptic vesicles from the electric organ of Torpedo marmorata by porous glass bead chromatography and estimation of their acetylcholine content, Exp. Brain Res., 24 (1976) 19. 14 Scha.ffner, W. and Weissmann, C., A rapid, sensitive and specific method for the determination of protein in dilute solution, Analyt. Biochem., 56 (1973) 502-514. 15 Sheridan, M. N., Whittaker, V. P. and Israel, M., The subcellular fractionation of the electric organ of Torpedo, Z. Zellforsch., 74 (1966) 281-307.
457 16 Stadler, H. and Whittaker, V. P., Identification of vesiculin as a glycoaminoglycan, Brain Research (1979) in press. 17 Stanley, P. E. and Williams, S. G., Use of liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme, Analyt. Biochem., 29 (1969) 381-392. 18 Tanford, C., The Hydrophobic Effect: Formation of Micelles and Biological Membranes, J. Wiley and Sons, New York, 1973. 19 Tashiro, T. and Stadler, H., Proteins of cholinergic synaptic vesicles of Torpedo marmorata, Europ. J. Biochem., (1979) in press. 20 Whittaker, V. P. and Barker, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, Methods Neurochem., 2 (1972) 1-52. 21 Whittaker, V. P., Essman, W. B. and Dowe, G. H. C., The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae, Biochem. J., 128 (1972) 833-846. 22 Whittaker, V. P., Dowdall, M. J., Dowe, G. H. C., Facino, R. M. and Scotto, J., Proteins of cholinergic synaptic vesicles from the electric organ of Torpedo: characterization of a low molecular weight acidic protein, Brain Research, 75 (1974) 115-131. 23 Wicksell, S. D., The corpuscle problem. A mathematical study o f a biometric problem, Biometrika, 17 (1925) 84-99. 24 Zimmermann, H. and Denston, C. R., Separation of vesicles of different functional states from the cholinergic synapses of the Torpedo electric organ, Neuroscience, 2 (1977) 715-730. 25 Zimmermann, A. and Dowdall, M. J., Vesicular storage and release of a false cholinergic transmitter (acetylpyrrolcholine) in the Torpedo electric organ, Neuroscience, 2 (1977) 731-739. 26 Zimmermann, H. and Whittaker, V. P., Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo: a combined biochemical, electrophysiological and morphological study, J. Neurochem., 22 (1974) 435-450.