Adenosine triphosphate in cholinergic vesicles isolated from the electric organ of electrophorus electricus

Bra& Research, 111 (1976) 365-376

365

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

A D E N O S I N E T R I P H O S P H A T E 1N C H O L I N E R G 1 C VESICLES I S O L A T E D F R O M T H E E L E C T R I C O R G A N OF ELECTROPHORUS ELECTRICUS

H. Z 1 M M E R M A N N and C. R. DENSTON

Abteilung fiir Neurochemie, Max-Planck-lnstitut fiir Biophysikalische Chemie, 3400 GSttingenNikolausberg ( G.F.R.) (Accepted December 5th, 1975)

SUMMARY

Synaptic vesicles have been isolated from the electric organ of the bony fish

Electrophorus electricus using sucrose step gradients and zonal centrifugation. Although the acetylcholine (ACh) content of the Electrophorus electric organ is only 2 ~ of that of Torpedo, ACh and A T P can readily be measured in the peak fractions using the leech microassay and the firefly luciferin luciferase assay respectively. The protein content of the vesicle fraction in experiments with Electrophorus was much higher than with Torpedo, but a possible contamination of this fraction with mitochondrial or cytoplasmic particles could be excluded. The ACh to A T P ratio of 10.8 is close to that found for cholinergic vesicles isolated from Torpedo and also to that of other amine storing granules.

INTRODUCTION

Isolation of synaptic vesicles from the electric organ of the Elasmobranch fish

Torpedo and Narcine, a tissue extremely rich in cholinergic nerve terminals, has been performed successfully several years ago 1,6,18. These vesicles contain acetylcholine (ACh) as well as adenine nucleotide. Most of this nucleotide could be shown to be present in the form of A T P 3,19. On electrical stimulation vesicular ACh and A T P decrease pari passu 23,24. Silinsky demonstrated the release of A T P from the rat neuromuscular junction on electrical stimulation 13. On the other hand it was impossible to release adenine nucleotides from the superior cervical ganglion of the cat 7, and it may be questioned whether A T P is a genuine constituent of all cholinergic vesicles as it is for other amine storing granules, e.g., chromaffin granules 5, dense-core vesicles from sympathetic neurones 1° or blood platelet granules 2. In order to obtain further information about the generality of the presence of A T P in cholinergic

366 vesicles we aimed to isolate synaptic vesicles from an animal further advanced in evolution. We chose the electric organ of the bony fish Electrophorus electricus since like its counterpart in 7bt7wdo it is a purely cholinergic tissue, although its ti.,sue content of ACh is much lower than that of the Torpedinidae. MATERIAL AND METHODS

Animals Fish (50-90 cm) were obtained from local dealers and kept in aquaria at room temperature. Animals were sacrificed by decapitation without prior discharge of their electric organs. Tissue was taken from the rostral end of the main electric organ.

Tissue fractionation All manipulations for tissue fractionation were done in a cold laboratory with a temperature of 0-4°C. Tissue was homogenized in a 'Waring' blender (3 or 4 times 30 sec at 12,000 rev/min with 30 sec intervals for cooling) in solutions (Vol -:: weight of tissue) of 100 m M NaCI, or 200 m M glycine or sucrose. Experiments in which tissue was frozen in liquid nitrogen and then pounded for homogenization did not give satisfactory results. The homogenate was spun in a Sorvall centrifuge at 16,000 × gm,~x for 30 min. The supernatant fraction (Sic) was loaded on a zonal gradient of increasing density of sucrose essentially as described by Zimmermann and Whittaker 24 (Beckman Ti-14 rotor, 115,000 x gav for 3 h). In some experiments free ATP in the parent fraction $12 was destroyed by addition of a mixture of apyrase and myokinase 3,24. In other experiments the $12 was centrifuged at 159,000 × g a v (50,000 rev/min in a Beckman AH 50 rotor) for 1 h to sediment vesicles and the supernatant (Sa) discarded. The pellet (Pa) was resuspended in isotonic medium, loaded on a step gradient with increasing density of sucrose (Fig. !) and centrifuged for 2 h at 58,000 × gay (21,000 rev/min in a Beckman SW27 rotor).

Analytical methods ACh was extracted from tissue with 10 ~/o trichloroacetic acid 24, from fractions with 0.1 N HC124 and assayed using the leech microbioassay method 17. The ATP determination was performed by the firefly luciferin luciferase assay t5 with minor modifications 24. Cholinesterase, lactate dehydrogenase, fumarase and protein in tissue extracts and subcellular fractions were estimated essentially as described by Whittaker and BarkerlL

Morphological methods The fixation, washing and osmication of all samples was carried out at 0-4°C. Whole tissue. Portions of tissue from electric organ were excised and fixed in 2 % glutaraldehyde, containing 0.05 M sodium phosphate buffer, pH 7.4 and 200 m M sucrose for 3 h. They were then washed in the above solution without glutaraldehyde for several hours and osmicated by immersion in 1% OsO4 made up in the buffer wash for 2 h. After dehydration in ethanol and embedding in Epon, thin

367

Fig. 1. Nerve terminals in the electric organ of Electrophorus in situ. a: low power electron micrcgraph of an electroplaque cell showing 3 nerve terminals (*) attached to the innervated face (if). The nucleus of the cell is surrounded by mitochondria and cisternae of the smooth endoplasmic reticulum. Filamentous structures are scattered throughout the cytoplasm. The basement membrane follows the infoldings (~) of the outer membrane of the electroplaque cell. s, interstitial space between electroplaque cells. Bar indicates 2 t~m. b: detail of synapse of an electroplaque cell. The nerve terminal is covered by a glial sheath (g). As well as synaptic vesicles, larger membrane sacs, partly with a concentrical double membrane can be seen. Some of the vesicles contain electron-dense granules (~). The presence of microtubules (*) indicates the close proximity of the axon. The basement membrane (m) continues throughout the synaptic cleft (c). e, postsynaptic electroplaque cell; s, interstitial space. Bar indicates 500 nm.

368 sections were cut with an LKB Ultratome-lll, stained with uranyl acetate and lead citrate and viewed in a Jeol 100 B electron microscope.

Subcellularfractions Different fixatives were used for vesicles in suspension. In each case the ice-cold fixative was added to 3 vol. of fraction. The final concentrations were as follows: fixative I" 1 ~,',{iglutaraldehyde, 20 m M sodium phosphate buffer, pH 7.2 60 m M sucrose; fixative II: as 1 but 25 m M sucrose; fixative 1II: as I but 12.5 m M NaCI instead of sucrose. After 1 h the fixed particles were sedimented at 105,000 ::, g (39,000 rev/min) in the SW 39 rotor of the Beckman ultracentrifuge for 2 h. The pellets were washed in buffer solution of the same composition as used for glutaraldehyde fixation for several hours, osmicated in 1 ° o OsO4 made up in the buffer wash for 3 h, dehydrated and embedded as for whole tissue. RESULTS

Homogenization It is apparent from Fig. 1 that the nerve terminals in the electric organ of Electrophorus are smaller and far less abundant than in the electric organ of Torpedo11. They are densely packed with synaptic vesicles, some of which contain a dense granule of about 15 nm diameter in the core. The vesicle diameter was calculated to be 57.4 ~: 0.8 nm (S.E.M., 66 vesicles measured) as compared with 84 nm of the synaptic vesicles in the cholinergic nerve terminals in the Torpedo electric organ ~z. The technique of tearing open nerve terminals by crushing tissue frozen in liquid nitrogen was succesfully used for isolation of vesicles from the Torpedo electric organ is. This procedure was not as satisfactory for preparation of vesicles from the organ of Electrophorus, probably because of the much lower density of innervation, Homogenization with a 'Waring' blender at high speed was found to be the most convenient method for direct isolation of synaptic vesicles from nerve terminals although about 50°,0' of tissue ACh activity is lost on homogenization. The mean A C h content (nmole/g) in the electric organs of 3 fish was found to be 17.5 ~:: 3.8 (S.E.M.). This is 1.9°o of the content found in Torpedo electric organs '~4. In one experiment the fate of the bound ACh was followed throughout the isolation procedure. The first homogenate contained 43.3 'Y0 of the tissue ACh content and the supernatant of the first spin ($12) 22.4°;~. When this supernatant was spun again for I h at 150,000 ,', gay in order to sediment synaptic vesicles, 3 9 ~ of the bound ACh stayed in the supernatant. As under identical conditions only about 10',',~ of vesicles isolated from the Torpedo electric organ would stay in the supernatant (Zimmermann unpublished) it can be concluded that the smaller synaptic vesicles from Electrophorus are less dense. No significant differences in the ACh content of fractions were obtained from tissue homogenized in either 100 m M NaC1 or 200 m M glycine or sucrose.

Subfractionation Step gradients. When the Pz fraction is loaded onto a gradient as shown in Fig.

369 1 2

5

5 O.2

m

m

5

10

7

8

8

Q4 O.6 .......

9

1.5

58 O00gx 2h ACh ATP (nmol)~ {nmm~[ )

Protein

Esterose

(Y)

[~'itslminl~"

'l"

,[,o 1

2

3

4

5

6

7 8 9 hr. of fraction

Fig. 2. Separation of vesicles isolated from the electric organ of Electrophorus glectricus by step gradient. Fraction P~ derived from 24 g tissue was loaded. Numbers in the tube give the vol (ml) of solution per gradient step, on the right hand side the molarity of sucrose and on the left hand side the identity (1 9) of the fractions collected; the small bars indicate the position of the cuts. Shading indicates bands. Units of enzyme activity as in Table II. Black area, acetylcho[ine; white area, ATP; hatched area: protein; stippled area, esterase.

1, 5 major bands are formed. Most of the ACh containing particles sediment in the band of 0.2 M sucrose. Another peak of ACh can be found in association with the heavy membranes (fraction 8) which is presumably due to synaptosome-like particles which were not completely destroyed on blending or due to incomplete resuspension of the pellet P3. ATP is relatively higher to ACh in fraction 2 than in 3 and 4, possibly indicating heterogeneity of the vesicle population. The content of protein and of total cholinesterase in the fractions containing vesicle bound ACh is rather high, thus indicating contamination with particles mainly derived from the innervated face of the electroplaque cell. In order to allow for possible contamination of the vesicle peak fraction with particles other than synaptic vesicles, the mean of ATP value of fractions 1 and 6 was subtracted as a background. Table I shows that the molar ratio of ACh to ATP of the peak tube in 4 experiments was 10.6 (7.0, without background subtraction). The ACh to protein ratio (nmole/mg) in 4 experiments was 3.5 (Table 1). The membrane fraction has by far the highest protein content. Table I1 gives the content of peak fraction of the various parameters measured per gram of original tissue as compared to the content in the parent fraction which was loaded on the gradient. Zonal gradients. Zonal centrifugation by continuous gradients leads to a high resolution of the various types of particles loaded. In all experiments where zonal centrifugation was used, $12 was loaded as the parent fraction. As shown in Fig. 3a there is a clear peak of ACh in the region between a density corresponding to 0.28 and 0.45 M sucrose. No ACh could be detected in the heavy membrane fraction. ATP

370 TABLE 1 The molar ratio o f ACb to ATP and the ACb to protein ratio in the vesicle peak/raction oj Llectrophorus electricus as compared to data obtained [i'om Torpedo marmorata and ox superior cervical ganglion

Values for vesicles from electric organs are means ± S.E.M. of (n) determinations. A Ch/A TP (nmole/nmole)

A Ch/prote#l ( nmole /mg

Electrophorus electricus

Step gradient extraction with 0.2 M Glycine Zonal gradient extraction with NaCI (0.1 M), glycine or sucrose (0.2 M), respectively

10.56 vh: 1.54:1 (4) 11.09 _-tz2.44

(6)

3.49 k} 0.67:1 16.06 z~-:3.03

(4) (5)

Torpedo marmorata

Zonal gradient continuous extraction with 0.2 M sucrose, 0.3 M NaCI Extraction with 0.4 M NaC1

5.32 + 0.45

(21)*

566 :~ 7 2 : 1 (4)** 1862.5 ~ 276.3:1 (17)§ 34t6.6 :+ 146.7:1 (5)§§

Ox superior cervical ganglion

Step gradient

--

3 to 14" * *

• Ref. 3. • * Ref. 18. • ** Ref. 16. § Values calculated from data published by Zimmermann and Whittaker 24. §§ Values calculated from data published by Zimmermann and Whittaker24 including only zonal runs where the vesicle peak was beyond fraction No. 30. The better separation from the soluble protein yields a much higher ACh/protein ratio. shows a small p e a k in the soluble fraction, a larger one c o i n c i d i n g with t h a t o f A C h a n d an even larger one in the h e a v y m e m b r a n e f r a c t i o n . A s the m e d i a n p e a k o f A T P exactly coincides with t h a t o f A C h , it is c o n c l u d e d t h a t the A T P is c o n t a i n e d in the s a m e particles as A C h . T r e a t m e n t o f the p a r e n t f r a c t i o n Slz with a p y r a s e - m y o k i n a s e leads to a large d i m i n u t i o n o f the soluble A T P , b u t n o t o f the A T P in the vesicle region a n d this therefore shows t h a t the l a t t e r A T P is occluded. A m o n g s t the m a r k e r enzymes tested in the s a m e e x p e r i m e n t (Fig. 3b), f u m a r a s e is c o n t a i n e d only in the soluble f r a c t i o n i n d i c a t i n g the absence o f m i t o c h o n d r i a l c o n t a m i n a t i o n in b o t h the vesicle a n d the h e a v y m e m b r a n e fraction. Lactate d e h y d r o g e n a s e ( L D H ) was det e r m i n e d before a n d after t r e a t m e n t o f the fractions with T r i t o n X-100. It can be seen t h a t by far the m o s t o f it is f o u n d in the soluble f r a c t i o n a n d t h a t a small a m o u n t o f b o u n d L D H (releasable by T r i t o n X-100) can be detected only in the h e a v y m e m b r a n e fraction. N o b o u n d L D H is detectable in the vesicle region. T o t a l esterase shows a d o u b l e p e a k c o r r e s p o n d i n g to the soluble a n d m e m b r a n e peak, b u t the activity in the vesicle region stays r a t h e r high i n d i c a t i n g c o n t a m i n a t i o n with f r a g m e n t s derived in p a r t f r o m the p o s t s y n a p t i c m e m b r a n e . The m o l a r r a t i o o f A C h to A T P is highest in the p e a k fraction (inset, Fig. 2), a p h e n o m e n a which can also be o b s e r v e d in T o r p e d o vesicles ~,4.

371 TABLE 11 Comparison o f the ACh, ATP, protein, esterase and fumarase content o f the peak fraction o f vesicles isolated from the electric" organ o f Electrophorus electricus

a: content ± S.E.M. (or range, if less than 2 experiments) (no. of experiments) of fraction of continuous zonal or discontinuous step gradient which corresponds to 1 g of original tissue, b: content S.E.M. (or range) (no. of experiments), of parent fraction which corresponds to 1 g of original tissue. Units of enzyme activity are change in extinction/rain (100 1 0 . D . ) at 340 nm (lactate dehydrogenase) 412 nm (esterase), 250 nm (fumarase), measured in a 3 ml, 1 cm cuvette.

a) Zonal gradient continuous

ACh (nmole/g)

ATP (nmole/g)

Protein (l~g/g or mg/g) *

Esterase (units/min "< g × 10 3)

Fumarase (units~rain x g)

LDH (units~rain x g)

0.27 ± 0.03 (6)

0.03 ± 0.006 (6)

21.47 ± 2.62 (5)

1.33 z:~0.33 (4)

0.37 ± 0.35 (2)

1.55

0.09 i 0.01

226.83 i 54.11 (4)

15.36 i 2.76 (4)

---

--

9.59 il.02(5)*

168.66 ~L14.86(4)

33.45 t-4.46(2)

--

3.02 69.13 0.35 (3)* i11.15(3)

Step gradient 0.50 ± 0.07 (4) b) Zonal(S12)

± 3.74 ±1.28(4)

Step gradient (P~) - -

i 0.80 (2)

9363.25 ± 1426.87 (2)

A s can be seen f r o m Fig. 3a, the A T P values do n o t reach zero on either side o f the vesicle peak. It is therefore difficult to exactly assess the c o n t r i b u t i o n to the m e a s u r e d A T P by the s y n a p t i c vesicles in the p e a k fraction. W e therefore used the m i n i m u m value o f vesicular A T P for o u r calculations which is o b t a i n e d by d r a w i n g a line t h r o u g h the lowest p o i n t s occurring before a n d after the peak. P r e s u m a b l y the actual vesicular A T P value is s o m e w h a t higher. As shown in Table I, the m o l a r r a t i o o f A C h to A T P was f o u n d to be 11.1 ( p e a k fractions o f 6 experiments). This is very similar to the r a t i o f o u n d in step g r a d i e n t f r a c t i o n a t i o n experiments. W i t h o u t b a c k g r o u n d s u b t r a c t i o n a r a t i o o f 5.3 was calculated. The m e a n o f the values f o u n d by the different p r e p a r a t i o n m e t h o d s w o u l d be 10.8. W i t h r e g a r d to protein, an i m p r o v e m e n t o f the s e p a r a t i o n c a n be o b s e r v e d : the r a t i o A C h / p r o t e i n ( n m o l e / m g ) is raised to 16.1, the highest value o b s e r v e d being 26. This is s o m e w h a t better t h a n r e p o r t e d for cholinergic vesicles isolated f r o m bovine s u p e r i o r cervical ganglion a6 b u t still very m u c h lower t h a n in vesicles isolated from the T o r p e d o electric o r g a n (ref. 18 a n d Table II). T h a t the s e p a r a t i o n is generally i m p r o v e d can also be seen from the values for p r o t e i n a n d esterase, as a b o u t 10 times less o f c o m p o n e n t per g r a m o f original tissue is recovered f r o m zonal p e a k fractions t h a n f r o m step g r a d i e n t p e a k tubes, a l t h o u g h m a t e r i a l l o a d e d o n t o the zonal g r a d i e n t was m o r e enriched in these substances. Vesicles were best preserved after fixation in fixative l I l c o n t a i n i n g NaC1 r a t h e r t h a n sucrose to raise the o s m o l a r i t y o f the buffer. In the fixatives c o n t a i n i n g sucrose

372 ACh I, TP m~l ] nmol , (%7--/

(b)

08 ACh ATP /

Fumorese

I

-. -\\

Protetn

LDH

Esterclse

f~'~slm~j C~-; ml "

,ums,'m~m f~c, E' - ~ . , r ~ /

4

I

05

Sucros~

~

L~n°l)

I I I I I

15

'

i:

!

l i g ~.l

I!

10-

l

t

o8

8-;

0.4

4

,

'.,

.... ......

,/e.2o

i

05-

i

i, iHo

:....

j

\ 0

2~o

/oo

O~

500

0

~310

" '

*

,

200

,

,

L

~,t,,~,~;,,

0

400

509

Vo; of grodlanf lrnH

Fig. 3. Zonal separation of vesicles isolated from the electric organ of Electrophorus electricus. Fraction $12 derived from 65 g tissue was loaded, a: concomitant peak of ACh and ATP at a density of 0.39 M sucrose. Inset: molar ratio of ACh to ATP over the peak region. ( ..... ) ACh, (-- . ) ATP, ( . . . . ) M sucrose, b: enzyme markers and protein (same experiment). (. . . . ) fumarase, ( . . . . . . . ) esterase, ( . . . . ) free LDH, ( t • 0 ) free plus occluded LDH, (. . . . ) protein. Note that appreciable amounts of occluded LDH are only found in the heavy membrane fraction. Values for LDH between fractions 1 and 22 are reduced by a factor 100. The arrow indicates the continuation of the curve at a higher resolution. Units of enzyme activity as in Table Ii.

the vesicular m e m b r a n e appeared to disintegrate. Fig. 4a shows the c o m p o s i t i o n o f the vesicle peak fraction. The synaptic vesicles are embedded in a matrix o f soluble protein which originates from the soluble peak. This c o n t a m i n a t i o n accounts for the low values o f the A C h to protein ratio. The small m e m b r a n e fragments --- sometimes o f the shape o f elongated synaptic vesicles - - probably add to the content o f cholinesterase in the vesicle peak region. The electron-dense granules can also be seen in the isolated vesicles, although Ca '-'~ was not present in the fixative, Similar dense granules have been observed in synaptic vesicles isolated from the Torpedo electric organ T M and are possibly due to the presence o f A T P 1. The m i c r o s o m e peak (Fig. 4b) consists m a i n l y o f heterogeneous m e m b r a n e fragments which are produced by the extensive blending o f the tissue. DISCUSSION

Tissuefractionation A c c o r d i n g to the fine structural analysis the electric organ o f Electrophorus is far less densely innervated than that o f the electric ray Torpedo 8,11. This is supported

373

Fig. 4. Morphology of fractions isolated by zonal centrifugation as shown in Fig. 3. Fixative 111. Bar indicates 0.5 Itm. a: vesicle peak fraction. The vesicles are embedded in a matrix of protein which seems to be derived from the soluble peak rather than from disintegrated particles. Besides spherical vesicles of the size found in whole tissue sections flattened profiles can be seen (*). Some of the vesicles contain an electron-dense granule ('l)- b: membrane peak fraction. The blending of the tissue produces a rather uniform mixture of membrane fragments of various shapes and sizes, presumably derived from all parts of the electric organ and whose origin cannot be identified in detail. The dense particles scattered between the membrane fragments could be glycogen.

by the finding t h a t the tissue c o n t e n t o f A C h in Electrophorus is o n l y a b o u t 2 I~',i o f t h a t in Torpedo. T h e r e f o r e the Torpedo e l e c t r i c o r g a n is o b v i o u s l y b e t t e r suited for b i o c h e m i c a l analysis o f the c h o l i n e r g i c n e r v e t e r m i n a l . O n the o t h e r h a n d a m o n g s t t h e s y s t e m s w h i c h are p u r e l y c h o l i n e r g i c , the n e u r o m u s c u l a r j u n c t i o n in v e r t e b r a t e s is e v e n p o o r e r in t r a n s m i t t e r c o n t e n t . T h e rat d i a p h r a g m ~1 c o n t a i n s no m o r e t h a n

374 10"/o of the ACh content of Electrophorus. In evolutionary terms the Electrophorus as a bony fish is remarkably more advanced than the Elasmobranch Torpedo. Homogenization of the tissue with a 'Waring'-type of blender is obviously better for breaking the synapses and releasing the vesicles than powdering of tissue frozen in liquid nitrogen - the method of choice with Torpedo tissue ~8. Although blending destroys about 50,~,,i of the initial tissue content of ACh, a large part of the preserved transmitter is still included in larger particles which are sedimented by the first spin and only 20-30 0,'0 of the initial ACh are loaded on the gradient for vesicle separation.

Protein content Although the protein content of the vesicle fraction is rather high, the analysis of marker enzymes shows that neither mitochondrial contamination nor particles containing occluded cytoplasm are present in this fraction. The only constituent of relative high activity is the esterase indicating the presence of contamination derived partly from soluble esterase and esterase bound to the innervated side of the electroplaque cell. Overlap from the protein content of the soluble fraction is another likely contribution, as the particles are sedimenting rather early in the gradient (peak fraction on zonal separation - - 20-23). Ultrastructural analysis shows, that the synaptic vesicles in Electrophorus are smaller than in Torpedo and in size rather resemble those of neuromuscular junctions. This might account partly for their lower ACh to protein ratio and for the fact that they are more difficult to sediment than Torpedo vesicles. In all respects the vesicles isolated by zonal centrifugation are purer than those isolated by step gradients. This is also true for the isolation of vesicles from the electric organ of Torpedo (Zimmermann unpublished). The ACh to protein ratio (up to 26 nmoles/mg) compares to that reported for vesicles isolated from bovine superior cervical ganglion (up to 14 nmoles/mg) I6, but it is much lower than for vesicles isolated from the electric organ of Torpedo (Table ll). Obviously the Torpedo vesicles are enriched to high purity with very little contaminating protein present in the fraction. The further the particles can be sedimented away from the soluble protein the higher the ACh to protein ratio becomes. The value of 3417 (nmoles/mg) found in our experiments compares very closely to that of the adrenaline containing granules isolated from chromaffin cells (3450 nmoles/mg) H. As the technique for the isolation of nerve trunk vesicles is improved, higher values are also reported for these organelles (up to 100 nmoles/nag) ~z.

A TP content Although the ACh concentration in the vesicle fraction is rather low (2-3'I~, of fractions obtained under similar conditions from TorpedoZ4), the firefly enzyme assay is sensitive enough to measure the ATP content. This method is not only very sensitive but also very specific for ATP ~5. The concentrations of the two components were somewhat higher in the vesicle fractions obtained from step gradients but this was impaired by a several-fold increase in protein. The molar ratio of 1 I. 1 of ACh to ATP for vesicles isolated by zonal centrifugation compares to the range of values

375 o b t a i n e d u n d e r basically identical c o n d i t i o n s for vesicles isolated f r o m the electric organ o f Torpedo 4. It is also very close to the ratios f o u n d for o t h e r a m i n e storing granules, e.g., chromaffin granules, granules isolated from s y m p a t h e t i c nerves of different tissues a n d b l o o d platelet granules. The values r e p o r t e d for these storage granules range between 1.3 a n d 12 (ref. 3). A T P is also a c o n s t i t u e n t o f p e p t i d e horm o n e c o n t a i n i n g granules f r o m the p o s t e r i o r p i t u i t a r y . The m o l a r ratio in this case ( v a s o p r e s s i n / A T P ) is s o m e w h a t higher (20.6) 9. The d a t a p r o v i d e d for Electrophorus" therefore f u r t h e r s u p p o r t the idea t h a t A T P might be a genuine c o n s t i t u e n t o f all cholinergic s y n a p t i c vesicles, a l t h o u g h evidence for the central nervous system is difficult to obtain. These vesicles are very similar to granules storing c a t e c h o l a m i n e s with regard to their A T P storage capacity. Close similarities between cholinergic vesicles a n d adrenergic granules have also been r e p o r t e d c o n c e r n i n g the c o n t e n t a n d c o m p o s i t i o n o f their core p r o t e i n s 2°. ACKNOWLEDGEMENTS W e t h a n k Dr. V. P. W h i t t a k e r for c o n t i n u o u s s u p p o r t and valuable discussion a n d Dr. M. J. D o w d a l l for help with the p r e p a r a t i o n o f the English typescript.

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