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

115

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

P R O T E I N S OF C H O L I N E R G I C SYNAPTIC VESICLES F R O M T H E E L E C T R I C O R G A N OF TORPEDO: C H A R A C T E R I Z A T I O N OF A LOW M O L E C U L A R W E I G H T ACIDIC P R O T E I N

V. P. WHITTAKER*, M. J. DOWDALL*, G. H. C. DOWE*, R. M. FACINO** ANDJ. SCOTTO Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW (Great Britain) and Department of Neurochemistry, Institute for Basic Research in Mental Retardation, Staten Island, N.Y. 10314 (U.S.A.)

(Accepted January 28th, 1974)

SUMMARY A study has been made, using gel filtration, of the membrane-bound and soluble proteins obtained when cholinergic synaptic vesicles isolated from the electric organ o f Torpedo marmorata and related species are exposed to water. The usual technique for releasing protein was to dialyze suspensions of vesicles separated by zonal centrifuging in distilled water; the freeze-dried residue was then filtered through columns of Bio-Gel A-5 m or Sephadex G-200. About 60 ~ o f the protein was recovered in a void volume fraction. On disc-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), this fraction gave 3 prominent high molecular weight components. Over 40 ~ of the protein was a low molecular weight, acidic protein which we have called vesiculin. Hydrolysates were rich in hydroxy amino acids as well as glutamate and aspartate. Vesiculin is loosely associated with a small molecule, probably a nucleotide or mixture of nucleotides. Estimates of molecular weight by amino acid analysis, disc-gel electrophoresis, gel filtration and sedimentation coefficient all indicate a value close to 10,000. Vesiculin differs in composition and molecular weight from other acidic brain proteins. It is suggested that vesiculin may constitute the counter-ion to acetylcholine within the vesicle core.

INTRODUCTION The isolation of cholinergic synaptic vesicles in milligram quantities from the * Present address: Abteilung for Neurochemie, Max-Planck-Institut for biophysikalische Chemie, D-3400 Gtittingen-Nikolausberg, Postfach 968, G.F.R. ** Present address: Istituto di Farmacologia e Farmacognosia della Universit~ di Pavia, Viale Taramelli 14, 27100 Pavia, Italy.

116

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electric organ of the Torpedo 22 has made possible a detailed examination of the structure and composition of these organelles. In this paper we describe the separation from the vesicle fraction of three membrane-bound proteins and a soluble protein, of molecular weight about 10,000, rich in acidic and hydroxy amino acids, and in loose association with a small molecule, probably a breakdown product of ATP. We have called this protein 'vesiculin' and suggest that it serves, in combination with nucleotide, as the counter-ion for acetylcholine. It differs in composition and molecular weight from chromogranin, neurophysin, tubulin and Sl00 protein, 4 other low molecular weight acidic proteins also present in nervous tissue. Preliminary accounts of this wock have been given20,21,e3,24. METHODS

Preparation of fractions Isolation of vesicles. Synaptic vesicles and soluble cytoplasmic proteins were isolated at 0-4 °C from electric tissue of Narcine brasiliensis, Torpedo marmorata and T. nobiliana by zonal centrifugation essentially as previously describedZL Density gradient fractions containing vesicles (VP) and soluble cytoplasmic proteins (SP) were collected and pooled. In some experiments vesicles were sedimented at 270,000 ~ g for 1 h along with larger membrane fragments (fraction P3) from the parent fraction (Sa2) before zonal centrifugation. Due to difficulties in the supply of live fish at the initiation of this work, much of the preliminary work was perforce done with tissue frozen immediately after collection and stored at --20 to --60 °C for some time before use; such tissue contained very little acetylcholine and the vesicles were isolated on the basis of their density. The basic findings were, however, confirmed on vesicle preparations from fresh tissue and the results relate to this unless otherwise stated. Dialysis. Fraction SP and VP (100-200 ml) were dialyzed at 0-4 °C against several changes of distilled water (4-5 l) for 60-70 h using a dialysis bag formed from Visking sausage casing by knotting at each end. After dialysis the contents of the dialysis bag were lyophilized. In some experiments sodium azide (0.1 ~) was added as an anti-bacterial agent; this made no difference to the results. Gelfiltration. This was carried out at 0-4 °C on Bio-Gel A-5 m (Bio-rad Laboratories, Richmond, Va., U.S.A.) (40ml), Sephadex G-200 and Sephadex G-50 (Pharmacia AB, Uppsala, Sweden) (120 or 50 ml), equilibrated before use with the appropriate solvent [0.4 M sucrose-0.2 M NaCI, 0.2 M NaC1 or 0.1 M KCI-0.05 M Tris-HCI buffer, pH 7.4 (see ref. 1) as indicated in the legends to the Figs.], and calibrated with Blue Dextran 2000, glutamate and lactate dehydrogenases, bovine plasma albumin, catalase, cytochrome C, bacitracin and ATP. The protein content and absorption at 260 or 264 nm of the eluant were determined. Enzyme markers were localized by measuring the enzyme activity of the eluant fractions: cytochrome C was localized by its absorption at 410 nm and bacitracin and ATP by their absorption at 250 nm. Analytical methods and characterization of components Analytical. Protein was estimated by the method of Lowry et al. 12 and total

CHOLINERGICSYNAPT1C VESICLEPROTEINS

117

phosphorus was determined in gel fractions by the method of Bartlett 4 scaled down to a total volume of 3 ml. Samples of protein were hydrolyzed for amino acid analysis by heating them with 6 N HC1 in a sealed tube under nitrogen for 17-24 h; the hydrolysates were kindly analyzed by Mr. D. Rassin using the method of Spackman et al. 17 and a Beckman Model 120C automatic amino acid analyzer. Determination of sedimentation coefficient. A preliminary measurement (at 20 °C) was kindly made by Dr. P. Johnson using a Beckman-Spinco Model E analytical ultracentrifuge and the artificial boundary method with Schlieren optics. The vesiculin sample was dialyzed against 0.2 M sodium phosphate buffer before the run. Disc-gel electrophoresis. This was carried out 19 on a 1 0 ~ w/v polyacrylamide gel containing 0.1 ~ w/v sodium dodecylsulfate, 6 M urea and 0.1 M sodium phosphate buffer, p H 7.4. Protein (150#g) was layered onto the gel in 2 ~ o w/v sodium dodecylsulfate, 8 M urea, 2 ~ w/v 2-mercaptoethanol, 0.005 ~ Bromophenol blue (tracking dye) and 0.02 M sodium phosphate buffer, p H 7.4. A drop o f glycerol was added to the protein layer to prevent mixing with a column of 0.02 M sodium phosphate buffer, p H 7.4, which was layered on top o f it and into which the upper electrode dipped. Electrophoresis was carried out at 5 m A for 4 h. After fixation of the protein in 50 ~ w/v trichloracetic acid it was stained by immersion in Coomassie Brilliant Blue, followed by destaining in 7 ~ acetic acid. We are grateful to Dr. R. J. T h o m p s o n for valuable advice on the technique.

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Fig. 1. Gel filtration of a crude electric organ synaptic vesicle preparation under hypo- and isoosmotic conditions. Gel filtration was through Bio-Gel A-5 m of fraction P8 from the electric organ of Torpedo nobiliana (frozen tissue) after suspension in (a) water, (b) 0.4 M sucrose-0.2 M NaCI. The column was equilibrated and eluted with 0.4 M sucrose-0.2 M NaCI and the aqueous suspension of fraction Pa was made 0.2 M with respect to NaC1 by the addition of the solid salt before filtration. The distribution of material absorbing at 264 nm in the effluent is plotted on the ordinate. In (a) the pellet was derived from 40 g of tissue, in (b) from 20 g; the results in (b) are plotted on twice the scale of (a) for comparison. (c): absorption spectra of peaks a a (O), b a (O) and aft (continuous line) showing characteristic absorption maximum of material in peak ft.

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118 RESULTS

Separation o f vesicle proteins into membrane-bound and soluble fractions Effect o f hypo-osmotic treatment on a crude synaptic vesicle J?action. Fig. l a shows that a suspension o f a crude vesicle pellet (fraction Ps) in water is resolved by gel filtration into 3 fractions: a turbid void volume fraction appearing between 10 and 25 ml and consisting o f m e m b r a n e fragments, a c o m p o n e n t (a) o f molecular weight approximately 40,000-50,000, and a second c o m p o n e n t (/3) o f molecular weight about 10,000. By contrast (Fig. lb), when fraction P3 was suspended in 0.4 M sucrose0.2 M NaC1, a medium iso-osmotic with elasmobranch plasma and o f equal density to synaptic vesicles, only the void volume and a-fractions made their appearance. \ 1,C

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Fig. 3. Absorption spectra of gel fractions from electric organ synaptic vesicles. An experiment similar to that shown in Fig. 2 (a), using vesicles isolated from Narcine electric tissue: (a) : release of vesiculin on dialysis and freeze-drying; (b): absorption spectra of FDVP peaks I and II. The distribution of protein in the fractions was measured by their extinction at 265 nm and expressed as a percentage of the total recovered extinction, and the runs were performed with 116/~g (VP) or 3640 #g (FDVP) of protein, using 0.4 M sucrose-0.2 M NaCi (VP) or 0.2 M NaCI (FDVP) as eluting medium. The material in peak a, like that in fraction S12 and the supernatant left after sedimenting the vesicles, had a pink tinge (2max = 410 nm) and since it was present in both suspensions, it presumably represents soluble protein present in fractions Sle that was trapped in the interstices of the pellet. Fraction $12 contains hemoglobin derived f r o m the blood present in the dissected tissue. The material in peak fl represents a component or components released by osmotic shock. This fraction differs (Fig. lc) in its UV absorption spectrum from the material in peak a and from most proteins in having a prominent shoulder or small peak in the region 250-260 nm, suggestive of the presence o f a nucleotide in the fraction. The absorption coefficient is also unusually high for a protein. Effect of dialysis and freeze-drying on a purified vesicle fraction. To establish that the material in fraction fl was present in synaptic vesicles we used vesicles separated from soluble protein and membrane fragments by zonal centrifugation. Due to the large volume of VP, sedimentation of the vesicles from it was impracticable, and to induce osmotic disruption recourse was made to dialysis against distilled water for 60-70 h. After dialysis, the contents o f the dialysis bag were freeze-dried and suspended in a small volume (ca. 5 ml) of 0.2 M NaC1 or 0.4 M NaC1 for gel filtration. The soluble protein (SP) fraction was similarly treated. The dialyzed and freeze-dried fractions from SP and VP are referred to as F D S P and FDVP. Figs. 2-5 show the results o f gel filtration, on Bio-Gel A-5 m, of F D V P and F D S P derived from various species; the elution patterns of VP and SP, the untreated vesicle and soluble protein fractions are given for comparison. It will be seen that the effect o f dialysis and freeze-drying on VP (Figs. 2a, 3a, 4a and b) was in each case to release an apparently identical low molecular weight component (peak II; vesiculin) having a molecular weight and absorption spectrum (Figs. 2c, 3b, 4c and 5d) similar to component fl in Fig. la. This material, and also the peak (I) of membranous ma-

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Fig. 4. Gel filtration of subcellular fractions from electric organ. Experiments were similar to that shown in Fig. 2 using vesicles and soluble protein isolated from T. nobiliana (frozen tissue). The distribution was expressed as in Fig. 3. (a): VP and SP; (b): separate experiment showing FDVP and FDSP; (c): absorption spectra of FDVP peaks I and II. Amounts of VP and SP equivalent to 2.1 and 0.66 g of tissue and amounts of FDVP and FDSP containing 190 and 1500/~g of protein were used for gel filtration. Calibration of the column was with bovine plasma albumin (BPA) (molecular weight 68,000) and cytochrome C (Cyt c). Elution on both runs was with 0.4 M sucrose-0.2 M NaCI. terial coming through in the void volume o f the gel, behaved (Fig. 5) as single components on refiltration. By contrast, the elution pattern o f SP (Fig. 2b) was relatively little changed by freeze-drying. There was sometimes a slight shift in the main peak (FDSP II) and this may have been due to the release o f vesiculin from vesicles present in SP; significantly, the void volume peak in SP (presumably representing vesicles contaminating the fraction) was reduced (Fig. 2b) by dialysis and freeze-drying. Aging, freezing and thawing or treatment with supersonic oscillations did not cause the release o f vesiculin from the isolated vesicles. For the large scale separation of vesicle proteins, Sephadex G-200 rather than Bio-Gel A-5 m was used, since faster running columns could be prepared from it and its resolution in the molecular weight range 10,000-50,000 is very much better. Absorption at 410 nm was again used as a marker for non-vesicular protein. Figs. 6 and 7 illustrate the elution patterns obtained from such preparations with a calibrated column o f Sephadex G-200. When F D S P was passed through the column

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Vol. of effluent (ml) Fig. 5. Gel filtration of vesicular protein subfractions. (a): gel filtration of FDVP derived from the Ti-15 zonal VP peak (stippled) shown in (b) and identified by its acetylcholine content. Each of the peaks, the void volume peak containing vesicle membranes (I) and the vesiculin peak (II), ran as a single component (c, I' and II' respectively) on refiltration in two superimposed separate experiments. (d): spectra of the two components showing the persistence of the absorption maximum at 260-265 nm of vesiculin. The preparations are from T. marrnorata. Elution was with 0.4 M sucrose~0.2 M NaCI. (Fig. 6, broken line) relatively little material came through in the void volume (peak I) and the main peak was a large soluble protein peak (IIa) which had a strong absorption at 410 nm. By contrast, F D V P (Fig. 6, continuous lines; Fig. 7) gave a large void volume peak I and a prominent vesiculin peak (lib) but little or no soluble protein corresponding to the main peak in Fig. 6 (broken line).

Characterization of the vesicle protein fractions About 40 ~o of the recovered vesicle protein from F D V P was vesiculin (Table I). In order to study this protein further, the vesiculin peaks from several column separations were pooled, dialyzed, freeze-dried and stored in sealed ampoules under nitrogen at - - 2 0 °C before use. Some loss occurred during dialysis, presumably because of the low molecular weight o f vesiculin. Absorption spectrum. Vesiculin has a strong absorption per unit weight of Lowry-positive protein (Figs. lc, 2c, 3b, 4c and 5d) with a peak in preparations from fresh tissue in the region 260-265 nm, but in stored material often at somewhat lower wavelengths. The molar extinction coefficient is 1.42-2.70 × 105 according to species (Table I) for a molecular weight o f 10,000, suggesting that vesiculin binds

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Fig. 6. Separation of protein subfractions on Sephadex G-200. Gel filtration, on Sephadex G-200, calibrated with lactate dehydrogenase (LDH), cytochrome c (Cyt c) and bacitracin (molecular weight 2400) (Bac), and equilibrated and eluted with 0.1 M KC1-0.05 M Tris-HC1 buffer, pH 7.4, of FDVP (continuous lines) and FDSP (broken line) prepared from frozen electric organ of T. nobiliana. Note separation of FDVP into two fractions, membrane fragments (peak I) and vesiculin (peak lib), which, on this gel, has a considerably higher retention volume than the soluble protein i n FDSP (peak IIa). E26o ( • ) , E41o (C)). a b o u t 8 molecules o f nucleotide/molecule. By contrast, the ultra-violet a b s o r p t i o n s p e c t r u m o f the void v o l u m e m a t e r i a l showed no p e a k o r s h o u l d e r at 260-265 n m (Figs. 2c, 3b, 4c a n d 5d). Disc-gel electrophoresis of vesicle proteins. Fig. 8 shows disc-gel e l e c t r o p h e r o -

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123

CHOLINERGIC SYNAPTIC VESICLE PROTEINS TABLE I YIELDS

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PREPARATIONS

Each line refers to a separate preparation of vesiculin made by gel filtration on Bio-Gel A-5 m (B) or Sephadex G-200 (S). Values in cols. 4-7 assume that vesiculin has a mol. wt of 104, that the protein moiety has an e2n0 of 1.2 x 10 ~ and the putative nucleotide an e2a0 of 1.54 x 104. Preparations from T. nobiliana utilized frozen tissue, those from T. marmorata, fresh tissue. Separation technique

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g r a m s o f the m e m b r a n e a n d vesiculin fractions s e p a r a t e d on S e p h a d e x G-200. T h e m e m b r a n e fraction (Fig. 8, left) is seen to c o n t a i n 3 p r o m i n e n t high m o l e c u l a r weight c o m p o n e n t s (Nos. l a , 2a a n d 4a) a n d 4 less p r o m i n e n t c o m p o n e n t s (Nos. 3a, 5a, 6a a n d 7a). By c o n t r a s t the vesiculin fraction (Fig. 8, right) has one m a i n low m o l e c u l a r weight c o m p o n e n t (No. 4b) a n d several m i n o r c o m p o n e n t s : c o m p a r i s o n with the e l e c t r o p h e r o g r a m o f the m e m b r a n e fraction shows t h a t one o f these (3b) is similar o r identical to one o f the c o m p o n e n t s o f this fraction (4a). C o m p o n e n t s 5b a n d 6b a p p e a r to be low m o l e c u l a r weight peptides, p e r h a p s b r e a k d o w n p r o d u c t s o f vesiculin. Presence o f phosphorus in vesicle protein fractions. P h o s p h o r u s was f o u n d to be present in b o t h the v o i d v o l u m e m e m b r a n e p e a k (Fig. 7, p e a k I) a n d the vesiculin p e a k (Fig. 7, p e a k IIb). A small a m o u n t o f c o n t a m i n a t i n g soluble p r o t e i n t h a t h a d diffused in f r o m SP (Fig. 7, p e a k I I a ) c o n t a i n e d no d e t e c t a b l e p h o s p h o r u s . T h e p h o s p h o r u s in p e a k I was largely (80 ~ ) s o l u b l e in c h l o r o f o r m - m e t h a n o l (2:1, v/v), i n d i c a t i n g t h a t p e a k I, as expected f r o m a m e m b r a n e fraction, was rich in p h o s p h o lipid. By c o n t r a s t over 809/o o f the p h o s p h o r u s in the vesiculin p e a k (IIb) was n o t soluble in c h l o r o f o r m - m e t h a n o l ; this is consistent with the presence o f nucleotide,

124

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Fig. 8. Disc gel electrophoresis of protein fractions. Fractions were from T. nobiliana and were run on a 1 0 ~ polyacrylamide gel containing 0.1 ~ sodium dodecyl sulfate and 6 M urea. For details see Methods. Left: void volume (vesicle membrane) fraction from Sephadex G-200 gel filtration of FDVP. Note the occurrence of 3 major slow running components. Right: vesiculin; note the occurrence of one major (4) and 5 minor (1, 2, 3, 5, 6) components.

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dialysis and freeze-drying. Note separation into two peaks, A (molecular weight ca. 20,000) and B (molecular weight < 10a). The small intermediate peak corresponds to undissociated vesiculin. Insert: absorption spectra of vesiculin A and B showing loss of peak at 260 nm from vesiculin A moiety, and retention by B moiety.

but does not exclude the possibility that some phosphorus is bound to serine or threonine. The amount o f phosphorus in 4 preparations from frozen tissue of T. nobiliana and one from fresh T. marmorata was rather variable, but corresponded, on average, to 2-3 molecules/molecule of putative nucleotide based on the absorption at 260 nm (Table I). The experiment with fresh T. marmorata tissue unfortunately gave a preparation of VP poorly separated from, and thus heavily contaminated with, soluble protein (and free nucleotide2Z); this probably accounted for the low apparent yield of vesiculin and the high apparent nucleotide content; nevertheless, the results provide general support for the findings with preparations from frozen tissue. Dissociation ofvesiculin. When vesiculin, isolated on Bio-Gel A-5 m or Sephadex G-200, was submitted to gel filtration on Sephadex G-50 (Fig. 9) the compound ran as two components which we have designated vesiculin A and vesiculin B. Vesiculin A contained Lowry-positive material and behaved like a compound of approximately twice the molecular weight of vesiculin (i.e. 20,000). Its absorption at 260-265 nm was greatly reduced. Vesiculin B contained little or no Lowry-positive material, behaved like a low molecular weight substance and showed an absorption maximum at 260 nm. This suggests that vesiculin binds a low molecular weight compound, presumably a nucleotide, and when this is separated by filtration on a gel that resolves components of molecular weight 10,000 from those of much lower molecular weight, the protein moiety of vesiculin changes its hydrodynamic properties, perhaps by dimerizing. In other work 24 vesicles have been found to contain ATP in a molar ratio with acetylcholine of about 1:10. Apart from a small amount of AMP, no other nucleotide has been detected in the vesicles; however, the tissue contains all

126

v . P . WHITTAKER e t a / .

the e n z y m e s required to convert A T P to I M P a n d h y p o x a n t h i n e . The variations in )-max o b s e r v e d with different samples o f vesiculin i s o l a t e d f r o m tissues t h a t had been s t o r e d for v a r y i n g lengths o f time w o u l d be consistent with p a r t i a l o r c o m p l e t e oxid a t i o n o f the a d e n i n e g r o u p to h y p o x a n t h i n e ; in the native state the nucleotide assoc i a t e d with vesiculin is p r e s u m e d to be largely A T P . Exchangeability o f vesiculin nucleotide. Since fraction SP contains large a m o u n t s o f free nucleotide, i n c l u d i n g A T P , experiments were carried o u t with [U-14C]ATP in a n a t t e m p t to find o u t w h e t h e r the nucleotide a p p a r e n t l y b o u n d to vesiculin a n d r e t a i n e d even after p r o l o n g e d dialysis c o u l d have resulted f r o m c o n t a m i n a t i o n o f the vesicle f r a c t i o n b y m a t e r i a l f r o m f r a c t i o n SP. [14C]ATP (0.5 # C i o f specific r a d i o a c t i v i t y 550 C i / m o l e ) was a d d e d to a fresh p r e p a r a t i o n o f vesicles c o n t a i n i n g , besides its own c o m p l e m e n t o f A T P , free A T P (10.9 n m o l e s / m l ) a n d o t h e r nucleotides derived f r o m the SP peak. Vesiculin was isolated after dialysis, freeze-drying a n d gel filtration o n S e p h a d e x G-200 in the usual way. A n a l y s i s o f the d i a l y s a t e a n d retentate for r a d i o a c t i v i t y showed t h a t dialysis for 60 h h a d r e m o v e d 84 ~ o f the r a d i o a c t i v i t y . F u r t h e r dialysis r e m o v e d only a b o u t 2 ~ o f the o r i g i n a l r a d i o a c t i v i t y , or 12.5 ~ o f w h a t r e m a i n e d after the first dialysis, s h o w i n g strong r e t e n t i o n o f p a r t o f the a d d e d [14C]ATP. Dialysis also resulted in the loss o f 54 ~ o f the a c i d - s o l u b l e m a t e r i a l a b s o r b i n g at 260 nm. It w o u l d thus seem t h a t

TABLE II AMINOACIDCOMPOSITIONOF VESICULINHYDROLYSATES For preparation of vesiculin samples see text. Hydrolysis was carried out in 6 N NCI for the following times: Samples I and II, 20 h; Sample IlI (a) 17 h, (b) 24 h. Amino acid

I

Ala Arg Asp Glu Gly His lie Leu Lys Orn Phe Pro Ser Thr Tyr Val Mol. wt.

Mean -3: S.E.M.

Mean Assumed no. composition of residues (moles~mole per molecule of tyrosine)

9.3 ~ 0.24 2.4 ~ 0.40 8.9 ~ 0.54 18.8 -5_:0.54 13.3 2_ 1.50 2.0 :~ 0.17 2.5 ± 0.02 4.5 ~ 0.55 3.9 ± 0.66 2.8 ± 0.67 2.0 4- 0.32 4.8 ~ 0.50 13.7 ~ 1.69 5.2 3_ 0.39 1.2 ± 0.20 4.1 ~ 0.49

7.7 2.0 7.4 15.7 11.1 1.7 2.1 3.7 3.2 2.3 1.7 4.0 11.4 4.3 1.0 3.4

Amino acid composition (molar %) of vesiculin samples

9.1 3.0 8.5 18.4 10.0 1.9 3.1 4.9 4.7 3.7 2.4 5.1 12.3 6.2 1.4 5.3

I1

9.4 1.2 7.6 17.5 17.2 2.0 2.1 2.9 2.1 4.2 1.1 3.4 18.6 5.2 0.6 3.9

HI (a)

(b)

8.9 2.8 10.0 19.6 12.6 2.4 2.6 5.2 4.6 1.7 2.2 5.7 11.6 4.4 1.3 4,3

10.0 2.5 9.6 19.8 13,3 1.6 2.2 5.1 4.1 1.6 2.5 5.1 12.3 4.8 1.4 2.9

--

8 2 7 16 11 2 2 4 3 2 2 4 11 4 1

3 9891

CHOLINERGIC SYNAPTIC VESICLE PROTEINS

127

most of the free nucleotides were lost during the initial dialysis but that about 16~o of this free nucleotide exchanged with nucleotide bound to protein. Following chromatography on Sephadex G-200 all the radioactivity was associated with the vesiculin peak showing that the nucleotide binding sites were not associated with the membrane fragments. Amino acid composition of vesiculin. Table II gives the amino acid composition of 3 samples of vesiculin. Samples I and II were both part of the same preparation of vesiculin isolated from an FDVP preparation from Narcine brasiliensis by gel filtration on Bio-Gel A-5 m (cf. Fig. 3a) and differed only in that sample II had been concentrated by a factor of 4 by ultrafiltration through an Amicon U2 filter which retains particles down to 103 daltons. Sample III was a sample of vesiculin prepared from frozen T. nobiliana electric tissue; it was separated from FDVP preparation by gel filtration on Sephadex G-200 (Fig. 6b) and dialyzed against 0.2 Mphosphate buffer, pH 7.4, before hydrolysis. It will be seen that the hydrolysates are rich in acidic and hydroxy amino acids and that the vesiculin samples from the two species have similar compositions. The presence of ornithine is surprising. Samples of GTP and ATP hydrolyzed under comparable conditions contained small quantities of this amino acid (or another ninhydrin-positive chromatographically identical material) together with several others, but in insufficient amounts to account for it on the basis of the putative nucleotide content of vesiculin (about 8 moles/mole). The small amount of vesiculin available and its strong absorption at 260 nm did not permit the determination of amide or tryptophan groups. However, when chromatographed on columns of the acidic ion exchange resin CM cellulose or the basic resin DEAE cellulose, vesiculin behaved like a strongly acidic protein. Thus, it passed through CM cellulose but was strongly absorbed onto DEAE cellulose; it was not eluted from the latter above pH 3.0. Assuming that the molecule contains one residue of tyrosine, the amino acid present in lowest concentration in the hydrolysate, the molecular weight of vesiculin is calculated to be about 10,000, in good agreement with the value from gel filtration. Examination in the analytical ultracentrifuge. In the artificial boundary cell, vesiculin gave a single, symmetrical and rapidly diffusing Schlieren peak. The sedimentation coefficient, Sw,20 was 1.1 :L 0.1 S at a concentration of 0.5 g/100 ml. For a partial specific volume, v, of 0.75 and a frictional ratio, f/f0, of 1.2 (typical values for a globular protein) this would correspond to a molecular weight of 8000. However, if the molecule were more elongated (f/f0 ~ 1.38), the molecular weight would be about 10,000, in close agreement with the value given by amino acid analysis and gel filtration. DISCUSSION

Vesiculin must be counted as yet another acidic protein of relatively low molecular weight occurring in nervous tissue, though as yet evidence is lacking as to how specific its localization is. Attempts to assay it immunochemically have failed,

128

V.P. WHITTAKER e t al.

TABLE III THE MOLECULARWEIGHTS AND AMINO ACID t~OMPOSITIONOF ACIDIC PROTEINS FROM NEURAL TISSUE Units: (a) amino acid residues/mole of protein: (b) moles ~ of amino acid. Values for chromogranin A are for ox adrenal medulla, but a similar soluble protein from adrenergic nerve vesicles crossreacts with anti-chromogranin A serum 7. For vesiculin, S100 protein, chromogranin A and neurophysin I, col. (b) has been calculated from col. (a). For tubulin, the values are those given in Table 2 of Olmsted et aL 14. Neurophysins II and C have compositions very similar to those of neurophysin 115. Protein Species Tissue Reference

Vesiculin Torpedo Electric organ Thispaper (Table 11col. 8)

$100 protein Ox Brain Moore 13

Tubulin Chromogranin A Mouse Ox Neuroblastoma Adrenal medulla Olmsted et al. 14 Smith and Winkler TM

Neurophysin 1 Ox Pituitary lobes Rauch et al. 1~

Mol. wt

9891

24,000

55,000

77,400

9367

(b)

(b)

(a)

(b)

(a)

(b)

6.4 1.6 11.2 1.6 19.2 4.8 4.3 3.2 9.0 9.0 2.1 . . 8.5 0.5 5.3 4.3 0.5 1.6 6.9

8.1 5.6 10.1 1.8 13.4 8.2 2.4 5.0 8.7 6.5 2.3 . 4.0 5.2 5.5 6.4 . 3.0 6.6

55 42 56 3 156 52 13 7 50 57 13

8.0 6.1 8.1 0.4 22.6 7.5 2.0 1.0 7.3 8.3 2.0 . 1.6 9.9 8.0 2.3 . 1.2 3.6

9 4 7 6 10 15 1 2 6 2 --

10.5 4.6 8.1 7.0 ! 1.6 17.4 1.2 2.3 7.0 2.3 --

3 9 6 2

3.5 10.5 7.0 2.3

1 3

1.2 3.5

AminoacM (a)

(b)

Ala Arg Asp Cys/2 Glu Gly His lie Leu Lys Met Orn Phe Pro Ser Thr Try Tyr Val

8 2 7 -16 11 2 2 4 3 -2 2 4 11 4 -1 3

9.3 2.4 8.9 -18.8 13.3 2.0 2.5 4.5 3.9 -2.8 2.0 4.8 13.7 5.2 -1.2 4. I

Total

82

(a)

12 3 21 3 36 9 8 6 17 17 4 . 16 1 10 8 1 3 13 188

.

.

. 11 68 55 19

.

.

. 8 25 690

86

b e c a u s e so far it has n o t b e e n p o s s i b l e to raise an a n t i b o d y to it 18. It a p p e a r s to be d i s t i n c t in b o t h m o l e c u l a r w e i g h t a n d a m i n o a c i d c o m p o s i t i o n f r o m o t h e r a c i d i c p r o t e i n s a l s o f o u n d in n e r v o u s tissue such as S100 p r o t e i n , t u b u l i n , n e u r o p h y s i n a n d c h r o m o g r a n i n A ( T a b l e III). I n m o l e c u l a r w e i g h t a n d a m i n o a c i d c o m p o s i t i o n it perhaps most closely resembles the neurophysins, which bind the neurohormones o x y t o c i n a n d v a s o p r e s s i n , b u t in its a s s o c i a t i o n w i t h a t r a n s m i t t e r a n d a ( p u t a t i v e ) n u c l e o t i d e a n d its t e n d e n c y to d o u b l e in a p p a r e n t m o l e c u l a r w e i g h t o n r e m o v a l o f t h e n u c l e o t i d e it a l s o s h o w s similarities to c h r o m o g r a n i n A. T h i s p r o t e i n , t h e m a i n s o l u b l e p r o t e i n o f c h r o m a f f i n granules8,16 a n d also o f n o r a d r e n a l i n e - c o n t a i n i n g n e r v e g r a n u l e s 2,5,7 l i k e w i s e exists in d i f f e r e n t states o f a g g r e g a t i o n a c c o r d i n g t o w h e t h e r A T P is p r e s e n t o r a b s e n t , A T P f a v o r i n g t h e m o n o m e r i c f o r m 9,1°.

CHOLINERGIC SYNAPTIC VESICLE PROTEINS

129

In preparation from fresh, unfrozen tissue there is very lime vesiculin in fractions other than those known by morphological examination to contain vesicles as their sole or main membranous organelle. However, this is not true for tissues that have been stored for many weeks at --20 to --60 °C. Morphological examination of such tissues shows that they still contain vesicles; these vesicles, though devoid of transmitter, can be isolated and from them vesiculin can be prepared, but the soluble protein (SP) fraction now may also contain variable but often significant amounts of a vesiculin-like protein which cannot always be accounted for by contamination with vesicles. Unlike that present in fraction VP, dialysis and freeze-drying is not required for the release of this protein. It is possible that the source of this material is vesicles that have broken down intracellularly during storage in the frozen state. Soluble protein specifically localized in cytoplasmic organelles can be expected to be present in soluble cytoplasmic fractions also if the organelles have broken down on storage or during isolation: an example from the literature is chromogranin A, a constituent of bovine splenic nerve granules, which is also found (47 ~) in the high speed supernatant of splenic nerve homogenates% The function of vesiculin in the cholinergic synaptic vesicle is not known, but one may speculate that it constitutes a relatively high molecular weight polyanion which serves as the counter-ion for the acetylcholine cation and so greatly slows the diffusion of the latter through the vesicle membrane. Interestingly, vesicles from T. m a r m o r a t a are estimated to contain 2z up to 680 nmoles of acetylcholine/mg of protein and about 58 ~ w/v of protein. If vesiculin accounts for about 36 ~ of the total protein of the vesicle (Table II) there must be at least 1900 nmoles of acetylcholine/mg of vesiculin, i.e. 19 moles of acetylcholine/mole of vesiculin of molecular weight 10,000. The amino acid analysis (Table III) indicates that one mole of vesiculin could have up to 23 acidic residues and 9 basic ones. With the contribution from the 8 moles/mole of putative nucleotide associated with the vesiculin, the net negative charge on the vesiculin-nucleotide complex might range from 22 to 38 equiv./mole of vesiculin depending on the proportion of nucleotide present as ATP in the intact vesicle. There are thus more than enough potential anionic groups provided by the vesiculinnucleotide complex to neutralize the charge on the acetylcholine cation stored in the vesicle. Furthermore, the acetylcholine-nucleotide-vesiculin complex would account for about 35 ~ w/v of the vesicle or about 63 ~ of the core. Supplies of vesiculin have not so far been sufficient to carry out binding studies with acetylcholine or its homologues. However, there is no reason to suppose that vesiculin specifically binds acetylcholine; indeed it is unlikely that there could be more than one or two specific binding sites/mole of protein. The ability of the vesicle selectively to store high concentrations of acetylcholine rather than other ions might be a function, not of vesiculin, but of a specific carrier in the vesicle membrane. Further, vesiculin might well bind other positively charged ions, including metals, and this might be the basis for the staining of cholinergic vesicles observed to occur with zinc iodide (P. G. Waser, discussion, p. 221, of paper by Barker et al.a). If acetylcholine is packaged as 'acetylcholine vesiculinate' then|the packaging resembles that of other pharmacologically active cations where there is also evidence

130

v . P . WHITTAKER el 6//.

for association with a polyanion. Thus histamine is associated with the acidic polysaccharide heparin in mast cell granules 11, and adrenaline and noradrenaline with the acidic protein chromogranin A in chromaffin and splenic nerve granules respectively. S100 protein and other acidic proteins in brain may well have similar functions with respect to other, as yet unidentified, transmitters. ACKNOWLEDGEMENTS

This work was supported, in part, by Grants No. G. 969/488/B from the U.K. Medical Research Council and B/SR/6474 from the U.K. Science Research Council (to V.P.W.). We are most grateful to Mr. D. Rassin for performing the amino acid analyses and to Dr. P. Johnson for carring out the analytical ultracentrifuge run, also to Mr. W. Bear, Mr. C. Denston, Miss F. Henderson and Miss H. Potter for skilled technical assistance. The continued interest of Professor F. G. Young and Dr. G. A. Jervis is gratefully acknowledged.

REFERENCES 1 ANDREWS, P., The gel filtration behaviour of proteins related to their molecular weights over a wide range, Biochem. J., 96 (1965) 595-606. 2 BANKS,P., HELLE, K. B., AND MAYOR, D., Evidence for the presence of a chromogranin-like protein in bovine splenic nerve granules, Molee. Pharmaeol., 5 (1969) 210-212. 3 BARKER,L. A., DOWDALL, M. J., ESSMAN,W. B., AND WHITTAKER,V. P., The compartmentation of acetylcholine in cholinergic nerve terminals. In E. HEILBRONN AND A. WINTER (Eds.), Drugs. and Cholinergic Mechanisms in the CNS, Almqvist and Wiksell, Stockholm: F6rsvarets forskningsanstalt, 1970, pp. 193-214. 4 BARTLETT, G . R . , Phosphorus assay in column chromatography, J. biol. Chem., 234 (1959) 466-468. 5 DE POTTER, W. P., DE SCHAEPDRYVER,A. R., MOERMAN,E. J., AND SMITH, A. D., Evidence for the release of vesicle proteins together with noradrenaline upon stimulation of the splenic nerve, J. Physiol. (Lond.), 204 (1969) 102P-103P. 6 DE POTTER, W.P., SMITH,A. D., AND DE SCHAEPDRYVER,A. F., Subcellular fractionation of splenic nerve: ATP chromogranin A and dopamine fl-hydroxylase in noradrenergic vesicles, Tissue and Cell, 2 (1970) 529-546. 7 GEFFEN, L.B., LIVETT, B. G., AND RUSH, R. A., Immunohistochemical localization of protein components of catecholamine storage granules, J. Physiol. (Lond.), 204 (1969) 593-605. 8 HELLE, K. B., Some chemical and physical properties of the soluble fraction of bovine adrenal chromaffin granules, Molec. Pharmacol., 2 (1966) 298-310. 9 HELLE, K. B., Properties of membrane-bound and water-soluble forms of chromogranin A and dopamine-fl-hydroxylase activity, Bioehim. biophys. Acta (Amst.), 245 (1971) 94-104. 10 HELLE, K. n., Studies ofchromogranin A, Dissertation, Universitetsforlaget, Bergen, 1971. 11 LAGUNOFF,D., Structural aspects of histamine binding: the mast cell granule. In U. S. YON EULER, S. ROSELL AND B. UvNgS (Eds.), Mechanisms of Release of Biogenic Amines, Pergamon Press, Oxford, 1965, pp. 79-94. 12 LOWRY, O.H., ROSEBROUGH, N.J., FARR, A. L., AND RANDALL, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 MOORE,B. W., Acidic proteins. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. 1, Plenum Press, New York, 1969, pp. 93-99. 14 OLMSTED,J. B., CAROLSON,K., KLEBE, R., RUDDLE, F., AND ROSENBAUM,J., Isolation of microtubular protein from cultured mouse neuroblastoma cells, Proc. nat. Acad. Sci. (Wash.), 65 (1970) 129-136.

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15 RAUCH, R., HOLLENBERG,M. D., AND HOPE, D. B., Isolation of a third bovine neurophysin, Biochem. J., 115 (1969) 473-479. 16 SMITH,A. D., ANDWINKLER,H., Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla, Biochem. J., 103 (1967) 483-492. 17 SPACKMAN,n. H., STEIN, W. H., AND MOORE, S., Automatic recording apparatus for use in the chromatography of amino acids, Analyt. Chem., 30 (1967) 1190--1206. 18 ULMAR, G., AND WHITTAKER, V. P., Immunochemical studies on cholinergic synaptic vesicles, J. Neurochem., 22 (1974) 452-455. 19 WEBER, K., ANn OSBORN, M., The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J. biol. Chem., 244 (1969) 4406-4412. 20 WHITTAKER,V. P., Origin and function of synaptic vesicles, Ann. N. Y. Acad. Sci., 183 (1971) 21-32. 21 WHITTAKER, V. P., La vdsicule cholinergique. In G. G. NAHAS, J. C. SALAMAGNE, P. VIARS ET G. VOURC'H (Eds.), Le Systdme Cholinergique en Anesthdsiologie et en R(animation, Librairie Arnette, Paris, 1972, pp. 45-60. 22 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. 23 WHITTAKER,V. P., DOWE, G. H. C., AND SCOTTO,J., Vesiculin: a possible counter-ion for acetylcholine in the cholinergic synaptic vesicle. In J. DOMONKOS, A. F. FONY6, I. HUSZ.~K AND J. SZENT.~GOTHAI,Abstr. 3rd int. Meet. int. Soc. Neurochem., Budapest, Akaddmiai Kiad6, Budapest, 1971, p. 266. 24 WHITTAKER,V. P., DOWDALL, M. J., ANn BOYNE, A. F., The storage and release of acetylcholine by cholinergic nerve terminals: recent results with non-mammalian preparations, Biochem. Soc. Syrup., 36 (1972) 49-68.