- Email: [email protected]
Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax
ARCHIVES
OP
BIOCHliiMISTIZY
Purification
and
AND
BIOPHYSICS
Molecular from
Properties Torpedo
M. E. ELDEFRAWI Section of Neurobiology
3@2-373 (1973)
169,
and Behavior,
of the
Receptor
Electroplax A. T. ELDEFRAWI
AND
Cornell
Received
Acetylcholine
May
L~niuersity,
lthaca,
Xew York 14860
17, 1973
The acetylcholine receptor of Torpedo electroplax is purified by aflinity adsorption using cobra toxin (,Vaja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 rnv benzoquinonium produces a protein that binds a-[1251]bungarotoxin but not [3H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 M carbamylcholine produces an acetylcholine receptor protein that binds [3H]acetylcholine, [“Hldecamethonium, [3H]nicotine, [‘“C]dimet)hyl-d-tubocurarine, and cY-[‘2SI]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine recept)or (as measured by acetylcholine binding) averaging 69.2 Z+Z14.6yc. The purity of t’he isolated acetylcholine receptor protein is estimated to be at best 87% , as judged by disc gel elect,rophoresis and electrofocusing. The purified acetylcholine receptor binds 7.X nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K, = 0.02 PM) and 5.1 nmoles a lower a&nit3 (KD = 1.97 PM). If the protein concentrat,ion used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations 77p to 10 PM. Binding of acetylor robra toxin and was partially choline was totally inhibited by a-bungarotoxin blocked by four nirotinic drugs, but, not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.
Isolation of acetylcholine (AcCh)’ rcceptors from electric organs of fish has advanced from the stage of identification of the correct macromolecule in subcellular preparations, ut’iliaing binding of reversible ligands or neurotoxins, to that of different degrees of purification (1-3). We have previously identified AcCh rrcept,or molecules in membrane preparations from the electric organ of the cl&ric ray, Torpedo marmorata, by virtue of the characteristics of its AcCh binding, which was reversible, of high affinity and blocked only by nicotinic ligands (4, 5). It 1 Abbreviation: a-bungarotoxin.
A&h,
acetylcholine;
cu-BGT,
also bound nicotirw, dwamethonium, and curare (6) and its concentration was equal t’o that reported for the wbungarotoxin (a-BGT) binding molcculcs in the samr tissue (7). We have also shown t,hat these recept,or molecules could be solubilizcd by various detergent,s and retain their drug specificity (8). Conventional biochemical techniques, e.g., gel chromatography, elect’rophoresis, electrofocusing, or den&y gradient centrifugation, have been camployed, wit’h limited success, in the purification of AcCh receptor (7-10). Their drawbacks were’ cithrr bhe low level of purification achieved or the 362
Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
PlU)PEltTIES
OF PUIZIFIEI)
denaturation of t’hc receptor. The highest degree of purity of a functional receptor reported was 6.3-fold, obtained by electrofocusing (10). The recent introduction of the technique of afinity chromatography (1 l13) had a gwat. impact on the progrckss of rwcptor purificat,ion. The method is bawd on the ability of protcGw to bind certain ligands rcverslbly and with high affinities, so that Ohe highcr t,h(l nffinit~y the desired protein has for the ligand that is coval~~ntl~linked to t,he solid support,, tho more prc,tt>in will bind strongly and the more nonspwifitally bound proteins \vill desorh by elution with buffers. Thr A&h rccept,or from clectric organs of Totpedo and the clrctric ccl, IClectrophorus electricus, haw bwn purificid to varying dcgrces by affinit,y Columbus (16 lS), but, thwc is no report yet, of the>isolation of a totally pure: AcCh receptor. The Sepharow-linked ligands uwd ww either quat’carnary ammonium compounds (lZ, 16, 18) or the specific nwrotoxin from cobra (14, 17). Wc have used a phenyl t.rimethylammonium ligand (19) but the purification of AcCh recaptor from Tel-pedo \vas only lo-fold and t,hc recovery was very Iow (Eldcfrawi, unpublished). In the present cbmmunication, we describe an affinity m&hod well adapted for large scale production of a highly purified AcCh receptor from Torpedo rlcctroplax. This receptor protein is identified by virtue of the characteristics of it,s binding of its t,ransmitter, AcCh as well as other rcwrsihltt ligands, and cu-XT. Also, thr prop&w of the purifictd A&h rowptor are compared to thaw of its mr~mbranc-bound or solubilizcd countwpart, and its amino acids arc analyzed. MATERIALS
AN11 METHOl)S
Mnterials. Source of the AcCh recept,or is the lyophilized pellet of 12,000y of l’orpedo n~~rmora~a electroplax, stored under nitrogen at -20°C (20). [3H]Acetylcholine (48 mCi/mmole) was obtained from New England Nuclear, and [‘%]dimethyl-dtubocurarine (119 mCi/mmole), [3H]nicot.ine (1.7 and ~Hjdecatnethonittm (67 mCi,’ Ci/mmole), mmole) from Amersham Searle. Their raditrchemical purity was periodically checked by chroof the krait Bu7/garu,T matography. Venoms lnullicinclus and the Thailand cobra Suj-n naje
A&h-RECEPTOR
363
.sia7,ce,,sis were purchased from the Miami Serpentarium. PuriJica~ion ad use of [he neurotositrs. a-Bungarotoxin was isolat,ed in pure form by electrofocusing followed by gel filtration on Sephadex G50, and its idetrt.ity checked by its blockade of [“H]AcCh binding by the l’orpedo AcCh receptor and by autoradiography on mouse diaphragms (21). It was iodinated with [12511 by Sepharosebound lactoperoxidase, as described for other proteins (22), to give 0.X Ci,‘mmole. The ncurotoxin from the cobra was purified, then covalently fixed to Sepharose -IS following the method of Karlsson el cri. (14). The concentration of toxin used in this reaction was 0.25 mg toxin per ml of packed gel. The affinity gel was stored at 4°C in the presence of 10-j M hierthiolate and t~setl within a mont,h. Isolatiot~ of A&h rereplor b!/ u.@r~if!l udsorp/io,/. For eacah run, 1.68 g of the lyophilized pellet of 12,OOOg (from 120 g of ?‘orperlo electroplax) was homogenized in 60 ml of a modified Krebs original Itinger phosphate buffer (23, 24) containing l(A Trit,on X-100. This fluid fraction was then shaken for 30 mitt at. 4°C and centrifuged at, 105,OOOgfor I hr. Fifty milliliters of the supertrata.nt. was added t,o 25 tnl of packed aflinity gel, which had been washed with 100 ml of the Krebs-Ringer buffer, and t,he mixture was stirred for 1 hr in a loo-ml beaker. It was then filt,ered on a glass filter and the gel, with the A&h receptor adsorbed, was l.ransferred back t,o the beaker. Fifty milliliters of Ringer with only 0.17; Trit.on was added, and the tnixt~ure stirred for 20 mitt, then filtered on the glass filter so as t,o remove light,ly adsorbed or nonadsorbed proteins. This step was repeated once more. Then 50 ml of 1 .M SaCl in Ringer containing U.Iyo Triton was added to the gel and stirred for 20 min before filtration, to remove nonspecifically bound proteins. This step was also repeated. To the gel, 25 ml of 1 M carbatnylcholine (Aldrich Chemical Co.) in Krebs-Ringer brtffer (withottt any Triton) was added, and the mixture stirred for 16 hr. Although the A&h receptor has a lower affinity for carbamylcholine than the cobra toxin, the high concentration (1 M) of carbatnylcholine is sufficaient to desorb A&h receptor from Ihe toxin which is covalently bound to the gel. All steps were carried out) at. room temperature. To collect AcCh receptor in the carbamylcholine solution, the mixture was filtered on a Millipore filtration assembly using 0.35 pm HAWP filt.er to eliminate any bacterial rontamination. The filtrat‘e was loaded int,o dialysis sacs and dialyzed extensively in several changes of 5 mx dibasic sodium phosphate (pH 7.4), until the concentration of carbamylcholine dropped below lO-‘O M. Protein was assayed by the Lowry method
364
15Ll~l5F1~AWI
ANl)
I51,l~I~:FI:AWl
after whirh the gels were stained for 2 -3 hr with (25), using as a standard crystalline bovine serum 0.2jf’; Coomassie brilliant blrlc and dcstainc~tl albumin from Sigma washed with ethanol and with 7(‘;, acetic* arid for several hol,rs. The gcll dialyzed against Tris-acetate-phosphate brlffer, was scanned at 570 nm in a l~e(~km:tn Acta I I I pH 7. Activity of AcCh esterase was assayed hy spec~trophotornctcr. the Ellman method (26) and carbohydrat,es by the ilnthrone procedure (27). I: I~:S[JIi TS, Rir,di,/g of cho/i//ergic liga,lrls. Binding of the reversible ligands was measured by equilibrium Vvo shall rwtrkt the uw of the term dialysis as previously descsribcd (5, 2X). In the (aase A&h rcwptor, throughout prowntation of of [aHlArCh, Tetrnm was added to the pllrified th(k Itwults, to the molccul~~sthat bind A&h receptor preparation and dialysis medirlm at 10 and show th(l prqwr pharmacological spcciand 1 ~1w, respectively, so as to totally inhibit fkity, and w shall quantit!atc: it according the minute amount, of AcCh esterase present (4). to tho numbw of A&h binding sites. Trypsin (EC 3.4.1.4) (Type 1) and a-chymotrppI’urz”licatior~ oj A c(lh receptor. The capacit) sin (EC 3.3.3.5) (Type l), both from bovine g(,l to wniovc: pancreas, are from Sigma. They were used at 1 of thr cobra t,oxin afiinity mg,‘O.2 mg of pllrified AcCh receptor in 1 ml, and A&h rcwptor from the: extra& of Torpedo incubation was for 1 hr at 23°C. Binding of ~1~ ckctroplax in Iiwbs original Ringer plus 1 ‘2 1’“5I]BGT was assayed by gel filtration on an X-in. Triton was dctcwnincd bv incubating etch Sephadex (+-SO rolumn with a void volume of 3.5 1 ml of p&cd gcll n-it,11dkfwcnt volun~cs of ml. The A&h receptor-a-B(;T complex elrrted in t,lw Triton clxt’ract ; thq aftw 1-hr incubathe void vollune, whereas fret a-B($T, render the tion, the affinity gc~l \vas romowd by filtraexact assay caondit,ions, eluted in 7-8 ml. Onetion and [:3H]AcCh binding to the filtrate hundred-nucroliter samples were t,aken from cac.h d(~tcwninc~d. FV:(bfound that 1 ml of paclwd fraction, and the radioactivity counted. gel cwuld adsorb 2.3 nmol~s of AcCh binding Amino acid c~~ral?/sis. The purified A&h recoupsitw u+cn it ~vas incubattrd with 5 ml of th(a acid at t,or was hydrolyzed in 6 N hydrochloric 110°C ill evacllated sealed tubes for 20 or 40 hr, Trit,on clxt,ract (Table 1). WP wlectrd the and subjected to amino acid analysis in a Beckman ratio of 2 ml of Triton oxtractS (-1mg protcki/ model 120C analyzer accaording to the method of ml) to 1 ml of pwlcc~d gc,l, so that, all A&h Moore and Stein (29). Analysis for cysteine was rcccptor prcwtwt, would adsorb to the gcsl. after subjection to performic acid oxidation a(:Ilwovwicw of protclin, A&h wt(wsc, and cording to the method of Hirs (30). Tryptophan iIcCh rwytor arc prcwntcd in Table II. was assayed spect,rophotometricallJT (31) and also, Bawd on pmoh~s AcG bound/mg protctin, by chromatography following p-toluenesrllfonic, A&h rcwptor uxs purified by this prowacid hydrolysis (32). To estimate the concentradurc: 25fold. In 13 runs, rwwwrks of an tion of Triton X-100 present in the prlrified AcCh A&h rCMq)tcJr actiw (i.c>., A&h-binding) recept,or, we compared its uv absorption at, 221 and 280 nm with a st,andard (nl1rve made with a constant conc*entration of bovine serum albllmin (0.2 mg/ml) and concent,rat ions of Triton varying from 0.005 to O.l“i;. EZrclrofoc,/csi,cg. The LKB Produkter column (110 ml rapacity) was used as previously described W-9. ‘,g; of AcCh “moles of nmoles of ml of extract A&h receptor AcCh Gel clec(rophoresis. The met,hod used was that (4 mg receptor adsorbed& receptor l)rotein/ml) of Davis (33), but only running gel was used, so remaining add& added to that there was no previous c.oncentration in a after 1 ml of spacer gel. One-hundred microliters of purified incuhationrL packed gel AcCh rereptor fraction containing 15 pg protein was diluted with an equal volume of 40% sucrose 0.664 0 100 1 blue added. and a drop of 0.059; bromophenol 100 1.328 0 2 This mixture was layered on top of a 7’;io acryl0.M !I7 1 ,992 3 amide-0.18’; bisacrylamide gel (pH X.9). A Buch2 Cifi 18 9.i 4 ler analytical disc gel elect.rophoresis was used, 50 8.1, 3 ,320 ,i __. and constant current of 1 mA/tube was applied at. tL AcCh receptor is herein llsed t,o mean the first,, then raised to 3 milltube aft,er the marking [“H]AcCh binding sites. dye had penetrated the gel. The rtln lasted 2 hr,
PI’,OPElLTIISS
OF PURIFIEL, TABLE
A&h-CECGPTOlt
II
Ii M;~VISKIKS OF PROTHIN, ACCH ESTIXLWC .IND A&H E:XIW~IMI~:ST ny AFFINITY ADSOIWTI~N Fraction
Protein me/ fraction
l’g
Triton in Ringer extract (50 ml) l’j;, Triton in Ringer extract after adsorption First, O.l$;, Triton in Ringer Second O.lP’; Triton in llinger First O.lc/;, Triton, 1 M ISaC in Ringer Second O.lcz Triton, 1 1% NaCl in Ringer A&h receptor fraction desorbed with 1 JI carImmylcholine in Ringer Total “; recovery .4verage rewveries experiments
of
15
I~IXICPTOR ORTMNICD IN ii PURIFIC.ITIOS TO THR NI,:UXOTOXIX GEL AcCh esterase
C” (’
365
A.Xa,n/ fraction
C’ ic
i\cCh receptor pmoles AcCh hound/mg protein at 1 Pi AcCh
c-c
140
100
x00
100
206
100
04
66
550
69
0
0
33 5.4
23.7 3.x
16X 54
21 6.7
0 0
0 0
2.2
1 .6
14.5
1.x
0
0
0.8
0.5i
3 .5
0.44
0
0
1.1
2.x
0.1
0.001
9x.5 100.45 Ik6.14
rangod hct~wcw 55 and X2 76, with an awrage of 69.%%. Thc~ affinity gcal I\-as recycled hi washing with 200 ml Ringer and rwwd, but rwovcrir~s of A&h rcwptor awraged only 40 %, \\hich might; indicatcb that a few AcCh rcwptor mol~culw rcmaiwd attnchcld to the gd. I)eso,ption by benxoyuinoniuin compared to ~at.banz~lcholirle. A Triton cbxtracstof Torpedo
5135
!)8.9 101.08 rtll.78 _____
72.5
72.5 60.2 fl4.6
not bind [3H]AcCh. To determine whether thcb abwnw of binding of cholincrgic drugs b>~ the hc~IIzo(~uinonium-dt~sorhrd fraction \vas dw to df>naturation of the rwq~tor, WC incubat8ed 1 5’; Triton extract of Torpedo c4cctroplax \vit’h 10 rn>r henzoyuinonium for 16 hr, thcln dialyzed out this ligand. X&h binding \vas decreased by 80 %. 1lLolecular properties
of the AcCtl
receptor.
c4wtroplax was add(bd to t8hc affinit’y gcbland Binding of [“H]AcCh to the purified X&h twatcbd as dcscribt~d ahow, but in t’hc final rcwptor ~vas mclasurcld ovrr the concentrastop, dworption was awwllplishcd by 10 rnx tion range’ from 5 no t’o 11 q. The Scatchard hc~rlzocluinonillrll insttbad of 1 RI carbamylplot is prcwntod in Fig. 2a, and demonstrates choline:. Aftw wduction of hcnzoquiIlonium the prwcnw of two afini&s (possibly reprcto a conwntration below 1 pnr by dialysis, scnting t)wo sites). Iterative analysis of the thcb fraction bound w[“~I]BGT as did th(x data by II311 360 computw givcu a high car~)am~lcholinct-dcworb(~d fraction (Fig. 1)) affinity component with a dissociation conbut th(b hcnzoquinonium frakon (having stant (Ku) of 0.0% I.~I and a maximum (‘onprotein qua1 to X0 ‘2 of that of thcx car- wntration ([B],,,,,) of 2.7 nmol~~s mg probamylcholinc: fraction) did not, bind AcCh, tAn, and a lo\\- affinity component with a ~iiwtirw, d(carnothoniurn, or dimc+hyl-rlK, of 1.97 PM and [B],,, of 5.1 mnolcs/mg tubocurarincb (Tabk III). III anoth(lr VX- protAn. Th(w numbers add to giw a total pckmc~nt,, aftc,r dworption by b~wzoquinonX&h-binding sitw of 7.8 nrnolcs smg proium, twat,mcbnt of the go1with 1 11carhamylt8c>in. Thcwc~ valuw arc’ based on protein choline as prwiously dwcribcd rwovcwd analysis by th(l Lowry method (25). When an additional 15 %, of thr protck, \vhich did protcin \v:ts wtimated from amino acid
360
ELDI’FRAWI
ANI)
f~:LDI’FRAWI TAL-11,IS I I I
lot
BINDING OF CHOLINEKGI(' I,I(:.\NDS I%?' Two I
Cholinergic
ligand
Binding by two preparations (pmoles bound/mg protein) “aihb$l$ desorbed
[3H]AcCh (1 /XVI) [14C]I>imethyI-d-tubocurarine (0.09 PM) [3H]Nicotine (1.4 PM) [3H]l)ecamethonium (I .4 PM) u-[‘251]Bungarotoxin
TABLR
Benzoquinonium desorbed
.i 13.‘, 425
0 0
1016 2370
0 0
2515
2.580
IV
BI,OCKM)E Froct,o” number
OF [“H]AcCH BINDING (arr 1 PM) TO PGRIFIED A&H RECEPTOR ny VARIOUS
CHOIJNEIWIC
FIG. 1. Gel filtration on Sephadex (i-50 of different fractions after incubation with ,-[1251]BGT. (.......) a-[1251]BGT alone; (A-A) original Triton ext,ract of Torpedo electroplax; carbamylrholine-desorbed fraction; (O-----O) (m - -m) benzoquinonium-desorbed fract,ion. Flow rate is 25 mI/hr, fraction volume is 1 ml.
analysis, its concentration in t’hc purified AcCh receptor fraction was 25 %I lower. Based on this lower prot’ein value, tho total specific [3H]AcCh binding in the purified AcCh receptor is calculated to bc 10.4 nmolcs/mg protein. The Hill plot of the data (Fig. 2b) gives a lincl wit’h a slope of 0.5 and the R, value (see Ref. 34) is calculated to be about 500. Incubation of the purified AcCh receptor with trypsin or ol-chymotrypsin for 1 hr at 23°C reduced the amount of [3H]AcCh bound at, 0.1 PM by 34.7 % and 66.5 %, rcspcctively. When binding of [3H]AcCh at 1 PM by the purified AcCh rwcptor \vas measured in t’hc presence of cholinergic drugs (100 PM) in t’he dialysis bath, the nicotinic OIWS blocked the binding from 42 to 77 %, but th(> muscarinic ones did not (Table IV). Addition of (Y-BGT or cobra ncut8rotoxin to the purified &4cCl1receptor rcsultod in t,otal blockade of Arch binding.
I)RUGS
.\ND
Cholinergic ligan& _____~~
NEUROTOXINS
% Blockade
Nicotine 77 d-Tubocurarinc 65 1)ecamethonium 43 Benxoquinonium 42 Pilocarpine 0 At,ropine 0 Cobra neurotosin 100 amBungarotosin 100 .~~ ~~~~~ ~.__ ~____. 11Each drllg was added to the dialysis bath at 100 PM, but each neurotoxin was added to the purified AcCh recept,or to give a final concent,rat,ion of 1 PM, then equilibrium dialysis was started 30 min later.
CM clcctrophorwis of the purified fract’ion, in the absence of SDS, showed that the protein did not precipitate and that 87% of it migrated as one band a little into the running gel (Fig. 3), which indicates a molecular weight abova 300,000. When a-[‘2”I]BGT was added to tho purified AcCh rwqtor and then rlrct’rophoresr:d without SDS, the radioactivity was rcst’rictcd t’o t’hat major band. Electrofocusing of the purified AcCh receptor shoLvc:d that -93 % of the protein had an isoelcct,ric point around 4.8 (Fig. 4). The purified AcCh rcwptor showed rcmarkable stability as judgc>d by its binding
PROPERTIES
OF PURIFIED
of [3H]AcCh. When stored at 4”C, it lost 10% of its binding after 14 days, but when stored at O”C, them was no loss up to thrett weeks. The concentration of Triton in the purified AcCh receptor fraction n-as calculatcd to be about 0.007 %,.
A&h-RECEPTOR
The amino acid analysis of the purified AcCh receptor is present,ed in Table V. The high concentra6ion of acidic amino acids reflects the acidic nature of the AcCh receptor. We found equal amounts of carbohydrates in the AcCh receptor preparation a.s
1I
-3
-2
367
-1
0
Log [Ll
FIG. 2. Binding of [‘H]AcCh hy the purified AcCh receptor. (a) Scakhard plot B. hmount, bound in pmoles/mg protein; I, = [“H]acet.ylcholine concent,ration in FM. (b) Hill plot of the data. B’ = [B]/[B],,,,, where [Blmax is obtained from the computer analysis of the Scatchard plot. [L] = [“H]AcCh concentrat,ion in PM.
36s
El
)EFltAWT
AN11 ELl)EFKAWI
in blanks of Ringer or bovine serum albumin processed through the affinity adsorption procedure. Thus, it, seems that the source of carbohydrates is the Sepharosc used in affinity adsorption, which is a polymerized dextran.
3. Scan of a gel st,ained with Coomassie FIG. Brilliant Blue after electrophoresis of the purified AcCh receptor.
FIG. 4. Electrefocusing cal density of the fractions at low pH (<3).
Identification of A&h receptor. Binding of (Y-BGT has become a favorite index for t,ht: identification of nicotinic A&h receptor (3, 7, 3.5, 36). Yet there arc: many observations that prow: t#he nonspecificity of cr-BGT, especially when used at high concentrations or in homogenates. We found that (u-BGT bound to membrane preparations from the axons of lobster walking legs, where no AcCh rcwptor is known to exist (37). Thwt also macromolcculcs cholinc~rgic-binding bound A&h and nicotine, but whereas AcCh differentiated br~twcctn thcsc axonic mwromolecules and tht: AcCh rccept,or in having a much lower affinity for tho former, nicotine and CV-BGT did not (37). It has also bwn reported that at high concc,ntrations, cu-BGT &a~bound to tho sarcoplasm of 111ous~~ phragm (36). These data, plus the present, finding that, b(lrizocluinoniulri possibl!- dcnat,uwd AcCh IYYx!~~t(Jr and dworbed :I protein that bound o(-BGT and not ,4&h or othw reversible cholinergio ligands (Table III), indicate that total dopcndcnw on binding of o(-BGT for idt4ficatitm of thca purified A&h receptor or calculat~ion of its dcgrec: of purity (15, 17, 18) is quite inade-
of purified AcCh recept,or using ampholyt,es (pH 3-10). Optiis measured at 2X0 nm after correction for the high absorption
quat(‘. WC, thtwforc~, still strew that. the binding is pharmacologically specific (5, S, 10). Aftor all, binding of insulin has bwn host index for AcCh rcwptor is its rwcrsibl(~ binding of its trwnsmittw X&h, under COIN- omploycd to identify the insulin rcwptor ditions and at concc~ntraticJns \vhwc: thus (11) and noradrenaline the p-adrcncrgic rewptor (3). T.4BLK
Lysine Histidine Arginine Aspart ic acid Threonine Serine (illIt amic acid Proline Glycine Alanine Half cyst,ine Valin? Met hionine Isoleucine Leucine Tgrosine I’henylalanine Tryptophan Ilexosamine
1
6.1 2.1 3 .i 11.x 6. :3 7.1 10.5 (i .2 Ii .4 6 0
4 .:I 2 3 .i .4 10.8 4 .:3 ti .!I 9 .4 8.1 7.7
2 .o .i .i
1.1 7 .o 3.0 :<.7 9.0 3.8 .i .:3 2.0 1 6
1.7 5.2 0 .3 3 6 4.4 2.111 06
.i ,i
A.fitlity adsorpiiott atrcl degree of purz’jication. The> abovo procedure of affinity ad-
4.8 2.1 .i 0
12.6 4.1 6.8 11.1 i 8.8 i.4 0.0 fi 0 1.3 4 .o 8.2 2.9 .5 1 -
4.G 2.3 .-I 2 13.1 4..i 6.8 10.4 .i .!J x.7 (i .2 1 .fi
7.1 2.i 3 .8 8.6 3 (i .i 3 2.0
1 :%
‘1 These values are calmdated from data on tolrlene-extracted AcCh esterase from E(ec/rophuUIS electroplax presented as pmoles per sample or the integers of amino acid residrles assuming four histidine residues per minimum molecular weight (;il), and t,hey are the average of a 24. and 2“-hr hJ-drolysis of the prot,ein. ‘1I;ach number is an average obtained from three different preparations from Ei;‘ler:lrophorras electroplax (.i2). The AcCh est,erase was either homogenized and t rypsin toluene-extracted, digested, or subjected to both treatments, followed by af6nit.y chromatography ’ These values are calculated from average data on 3 samples presented as residues per 12 valinc residues. Acetylcholine esterase 11sed was tolllene-extracted from Elw/rophorus electroplax and purified by afhnit y chromatography (.X3). ,’ This value was determined by two met hods (81, 32). r We found 1 mole of carbohydrates per 10,000 daIt,ons of prot,ein. An equal concentration was found in a blank (I$; Triton in Uinger) and in a bovine serum albumin preparat,ion, both srlhjected to the same affinity adsorption prorcdllre.
sorption apparclntly offers scwral ndvantagcis owr the USCof affinity cwlumns. First is tlic, fact that it is not limited to the size or rat(> of flow of a colunm. WV haw purificbd Z-10 mg of AcCh rcwptor in a single run, and it is possible to process up to 1 g of A&h rcwptor. The awragc rccowry value of 69.2 “;) (T&1(, II) is the highest yet rcportcd, eompawd to 30-50 (l:‘,), 55 (18), and 60 !‘; (l(i), valuw bawd on LU-BGT binding. WV haw purified the XcCh rcccptor and cLnd(bdI\-ith 7.S to 10.4 nmol~~sof AcCh-binding sitca pw mg prot’cin, as judgc>d by sprxrific A&h binding (Fig. 2, Tabk IV), which arhiww a %-fold purification by afFinit> adsorption. Thrsc valurs may bc compawd with 2.7 and 6 nmolrs cu-BGT-binding sitwl mg protck from electric organs of narcintl and Torpedo califortrica2, rrspcctiwly, quivalcnt to 13.9 and 2%fold purification. Thcscl results \vcr(’ obtained by Schmidt and Raftclry using affinity chromatography on cowtlcwtly Ii&cd quaternary ammonium ligands ;t~l clution with NaCIkSa phosphate l)uff(br (16, IS). I
370
ELDEFRAWI
AND
2-3.3 nmoles/mg protein based on ol-BGT binding, and the multiple of purification by the affinity chromatography step was 130fold. The purification multiple that’ is theoretically feasible to obtain a pure AcCh receptor depends on t’he original concentrat,ion of AcCh receptor in the tissue. For example, we have shown by reversible binding of cholinergic ligands that the concentration of AcCh rcceptor in Electrophorus electroplax was 0.02-0.03 nmoles/g tissue (39), compared to a concentration of 1 nmoles/g of Torpedo electroplax (5, 20). Similar values mere obtained using a-BGT (7), cobra t,oxin (40), or maleimide compounds (41). Since the concentration of protein/gram tissue is similar in the two electric organs (20, 39), to arrive at an equally pure AcCh receptor requires 2030-fold more purification from the eel than does that from Torpedo. The purification that we have presently achieved is 2.5fold by affinity adsorption, Z-fold through membrane preparatjion (20), and 2-fold through Triton solubilization (S), adding to a total of 100-fold purification. Based on prot’ein and ol-BGT distribution in elcctrophoresis (Fig. 3) and the electrofocusing data (Fig. 4), it seems that the AcCh receptor obtained is, at best, 87% pure. This is still higher than everything so far reported on AcCh receptor of Torpedo californica or T. marmorata, as evidenced by the multiplicity of prot,ein bands in clectrophoresis or electrofocusing, respectively (18, 14). Total purificat,ion of AcCh receptor of Torpedo may thus require a II&fold lcvcl; accordingly, tot’al purification of AcCh receptor of Electrophorus electroplax would require a tot’al of 2300-3450-fold. Thcrrfore, the tot’al degree of purification so far achieved for Electrophorus AcCh recrptor, given at 1300-fold with a-BGT binding of 2-3.3 nmolcs mg protein (15), wprcsents only 40-60 % purification. Properties of the puri$ed AcCh receptor. This purified AcCh receptor still binds [3H]AcCh with two high afkitiw (Fig. 2a) as it did when at’tached to membranes (5) or after solubilization (8). The ratio of the low to high affinities is changed from 1.9 to 2.25 t’o S.3 in the purified, Lubrol-solubilizcd, and membrane-bound AcCh receptor, respec-
ELDEFRAWI
tively. Similarly, the Hill wefhcient is changed from 0.98 (5) to 0.66 (S) to 0.5 (Fig. 2b) in the three preparations, rospcctively. A Hill coefficient of 0.5 and an R,qof 500 for the purified AcCh receptor arc indications of ncgat’ivc: cooperativit!. (34, 42). Desensitization has been obwrvcd in electrophysiological expclriments in musclw and Torpedo cblectroplax (43, 44), whrrc exwss agonist conwntration kd to a reduct’ion in response, a phwomcwon which may bc considered as a case of ncgativc cooperat’ivity. Positive coopwativity has also bwn obwrvcd in a monoccWar preparation of WI c,lwtr+ plax, whore a Hill rorffick!~t~ of closc~to 2 \vas obtained with carbamvlcholitw (1.5). Such physiological responses arc tht> end result of a number of molecular cwnts t,hat, start’ with binding of a ligand to tlw A&h rcwpt.or. Therefore, tho clectrophysiological observations might, bo refkct~ions of events at the ligand binding step or subseqwnt OIIW. Our present observations suggest that, ncgat)ivc coopcra0ivity m:ty occur at th(> binding step. Although WChave not obscrvcd positive cooperativity in thr binding of AcCh to the purified A&h rweptor, it is possible that it may bc detected if binding is studied at low conccntrat’ions of [“H]AcCh which increaw at very small incremc:nt’s3. A single protein such as AcCh receptor may well clxhibit both positiw and ncgativc coopcrativity, depending upon the part’ of the ligand concentration curve under cxaminat’ion (46). When cu-BGT binding was studkd to ~lwkoplax of T. califorrzica, what may bcl t\vo sites was also found (47). Kot only do th(h conc&rat,ions of binding sit,w differ in thch purified AcCh rwxptor, but thcbaffinitks also decrease about IO-fold owr thca Lubrol-solubilizcd AcCh rcwpt~or. Anoth(ar change is the absence of aut,oinhihition up to 11 ~LJTA&h, t,hough n-c’ had ohswwd it abow 1 pc1-\1 in the mrmbranc prcbparat’ion (4X) and also th(> solubilizcd preparation, by c~quilibrium dialysis or by ultrafiltration (49). This phonomrnon was not, dw to wnctIivation of AcCh 3 After submitting this rnanusrript, we det,ected positive cooperativit,y it) rlrCh billding to this purified AcCh recept,or preparation at, i\cCh COIIcentrat,ions ranging from 0.0007 t,o 0.01 &l (Eldefrawi, M. E., and Eldefrnwi, A. T., Rioche/w. Pharntacol. Rep. Cotuv~. 1 (in press)).
PROPERTIES
OF PURIFIED
&erase, because we did not detect any increase in the concentration of acet’ate after dialysis at high concentrations of AcCh (Eldefrawi, unpublished). It, may be Ohat a polypeptide or a phospholipid responsible for t,hc aut’oinhibition was lost during purificaDion, or it is possible that the phenomenon may still occur hut’ at higher AcCh concentrations,. which \vould make it undet)ectable by cqmhbrium dialysis. On the other hand, the receptor protein is st.ill highly acidic wit,h an isorlcct,ric point of 4.S, which is not significantly different from t,hat of 4.5 f 0.2 we reportcld for the solubilizcd AcCh receptor (10). An interesting obscrvat#ion is t’he difference in binding of cholinergic ligands by the protein dcsorbed by carbamylcholine as compared to benzoquinonium (Fig. 1, Table III). The lack of binding of reversible ligands by t#h(abrnzoquinonium-dcsorbed protein may be due to a denat’uration possibly caused by an impurity present in the bcnzoquinonium solution or by the long exposure to the high concentration (10 m>r) of benzoquinonium used. Another alternat’ive is the incomplete removal of bcnzoquinonium by t,he extensive dialysis, which seems to bc a rem& possibility in view of the reversible nature of the in situ effect of bcnzoquinonium (50). To explain binding of LY-BGT in the presence of benzoquinonium, Tve would have to assume &her that a-BGT has separate binding sites from those of the reversible cholincrgic ligands or that the bulkier wBGT forms a larger number of bonds w&h AcCh receptor overlapping the areas of binding sites of the reversible cholinergic ligands. If one assumes that the Torpedo AcCh receptor is 57 % pure at the specific activity of 7.8 nmoles/mg protein (based on Lowry analysis) or 10.4 nmoles/mg protclin (based on amino acid analysis), then the molecular lvcight of t’hc subunit, which binds AcCh would be in the range of 83,000 to 112,000 daltons. Determination of the sizes of the subunit’s after different chemical trratmcnts, and following them by clcctrophorcsis, dynamic sedimentation, and electron microscopy, is under current investigation. Electron micrographs of the negatively stained purified AcCh receptor show doughnutshaped globular molecules (Salp&er and
A&h-RECEPTOR
371
Eldefrawi, unpublished). This highly acidic AcCh receptor protein has a high tendency to aggregate as we have previously reported (8, 20), which may indicate that there are areas of high densities of positive charge in the molecule despit’e its overall negativity. The degree of aggregation varies under different concentrations of salt or pH. The binding of [3H]AcCh indicates the presence of two affinit’ies (Fig. 2a), which may be due to two distinct sites on the molecule, two conformational states, or possibly different degrees of aggregation. The binding constants may change depending on the degree of aggregation and the detergent present, and thus may not reflect the in tivo situation. Many similarities were found between AcCh receptor and AcCh e&erase (e.g., presence in the same tissue fractions, similar concentrations, binding of AcCh, and comparable effects of chrmical reagents) that led to the earlier suggestions that the two are ident,ical macromolecules (see Ref. 1). However, diffcrmt purificat,ion methods have SUCceeded in partly separating these two macromolecules, and affinit’y chromatography was by far the best (14, 15, 18). In our purified AcCh receptor fraction, we calculate only one catalyt#ic site of A&h esterase still present’ for every 20,000 AcCh binding sites. There appears to bc more similarities than t,hcrc are differences in the amino acid content bct#ween AcCh receptor and A&h esterase (Table V). Thr basic amino acids make up approximately 12 mole % of either macromolecule, but AcCh rcccptor has about 33 % more lgsinc and 33 % less argininc than AcCh esterase. The acidic amino acids make up about 20-23 mole %, of either macromol~~culc, with approximately equal representation by aspartic and glut,amic acids. Acetylcholinc receptor has a higher molt % of t#hrclonine, half cystine, and isolcucine, and less of glycinc and valine. It stems that the source of carbohydrates in the purified AcCh receptor fraction is the Sepharosc used in affinity adsorption. It is still possible that a hexosaminc may be a component of the AcCh receptor molecule, but it,s presc’ncc might br: masked by t’he large amounts of carbohydrates in the fraction. By comparison, two of the analysrs of A&h esterase have reported the prrsrnce of hexosamine (51, 52). It would h(a more
372
ELDEFRAWI
.4X1) ELDEFRAWI
relevant to compare the two molecules from the same species, but unfortunately the amino acid analysis of Torpedo AcCh esterasc is not available. The reduction in [3H]AcCl~ binding observed after incubation of the purified AcCh receptor with trypsin or a-chymotrypsin poirks to the possible importance of the peptide bonds involving the carboxyl groups of arginine or lysine and tyrosine or phenylalanine for the activity of the AcCh receptor. The previously observed effect of 1,4-dithiothreitol in reducing AcCh binding by the membrane-bound AcCh recept#or (10) points to the importance of the disulfide bonds to AcCh receptor activity. These various bonds, and possibly others, are apparently essential for preserving the appropriate conformation of the AcCh receptor necessary for its binding of AcCh, and their relationship to the active site is yet to be elucidated. ACKNOWLEDGMENT We thank Mr. N. Balderrama for help in t,he electrophoresis experiments and Ms. S. Kane for technical assistance. We are grateful to Professor D. Wilson, Cornell University, for the time he spent in the analysis of amino acids and helpful discussions; and Professor T. C. Vannaman, Duke University Medical Center, for the analyses of tryptophan. We are also grateful to Professor R. D. O’Brien, Cornell University, for providing the lyophilized Torpedo electroplax used in the present research, which he collected at the Zoological Station, Naples, Italy; and the benzoquinonium sample presently used, which he received as a gift from Professor G. Webb, University of Vermont at Burlington. This research was partly financed by National Institute of Healt,h Grant No. NS-09144. REFERENCES 1. O’BRII~:N,
R.
D., ELDPFRA~I, M. E., SND A. T. (1972) Annu. Rev. Pharmacoz. 12, 19. 2. HALL, Z. W. (1972) Anuu. Rev. Biochem. 41, 925. 3. POTTER, L. T., AND MOLINOFF, P. B. (1972) i,l Perspectives in Neuropharmacology, (Snyder, S. H., ed.) p. 9, Oxford University Press, London. ELDF;FR~WI,
4. ELDEFRawI,
M.
E.,
BRlTT&:N,
A.
G.,
AND
O’BRIEN, R. D. (1971) Pestic. Biochem. Physiol. 1, 101. 5. ELDEFR~WI, M. E., BRITTIGN, A. G., *~.IND ELDRFR.ITVI, A. T. (1971) Sciejlce 1’73, 338.
6. ELDEFR.\\VI, MOUIt,
L.
M. P.,
I<., b:LDI:FR.\\\.I, .~ND O’BI~IKN,
A.
I:.
T.,
(;lI,-
1). (1971)
Mol. Pharmacol. 7, 420. 7. MIL~XJ, IL., MOJ~INOFF, P., .~ND POTT~;R, L. T. (1971) :Vaturc: (Lodo~) 229, 554. 8. ELJ)EFR.Z~I, M. E., I?LJ)I,:FK.\~vI, A. T., SXIFliJtT, S., .\ND ()‘BKII,:N, 1:. 1). (1972) Arch. Biochem. Biophys. 160, 210. 9. MEUSIXI~, J.-C., OLSEN, 1:. W., R/Ib:N,CZ, P., B. C., Elliott, W. H. and Jones, K. M., eds.), p. 208, Oxford University Press, Oxford. I<. I). (1970) 24. ELDJXFR~IWI, A. T., .~NI) O’BRIEN, J. Zeurochem. 17, 1287. 25. LOWRY, 0. H., I~OSIGRROUGH, N. J., F~RR, A. I,., AND IZIND.\J,J,, I<. J. (1951) J. Rid. Chem. 193, 265. G. L., COURTNET, K. D., ANI)EKS, V. 26. ELLM:\N, JR., ~NJ, FR:ITHI;RSTONI.:, IL. M. (1961) Binthem. l’harnaacol. 7, 88. 27. COLOWICK, S. P., .4x1) K.~PL:YN, N. 0. (1957) Methods E~zymol. 3, 73. 28. ELDJGFR.\WI,
M.
E.,
EJ~DCFK.4w1,
A.
T.,
.\ND
PROPERTIES O’BRIEN, 104.
OF PURIFIED
R. D. (1971) Mol. Ph,armacol. 7,
29. MOORE, S., AND STEIN, W. H. (1954) J. Riol. Chem. 211, 893. 30. HITS, C. W. H. (1956) J. Biol. Chem. 219,611. 31. BENCZF,, W. L., >\ND SCHMID, K. (1957) A,la!. Chem. 29, 1193. 32. Lru, T.-Y. (1972) Methods Evzymol. 26, 44. 33. I)AVIS, B. 12. (1964) L~WL N.Y. .I&. ,%i. 121, 404. 34. KOSHLAND, I). E., JR., N~METHY, G., .z~u FILMF,IL, D. (1966) Biochebstry 6, 365. 35. STY-CKOWSKI, A, J., VOGEL, Z., AND NIREKHKRG, N. W. (1973) Proc. Sat. Acad. Sci.
USA 70, 270. 36. PORTER, C. W., CHIN, T. H., WIHXO~SKI, J., AND BARNARD, E. A. (1973) &V\‘atxre *Vew Riol. 241, 3. 37. DICNBURG, J. L., ELUXFRA~I, ?vl. E., AND O’BRIE:N, R. D. (1972) Proc. Sat. Acarl. Sci. USA 69, 177. 38. LEFKOWITZ, It. J., .\ND I%ABER, E. (1971) Proc. Nat. Acad. Sci. USA 68, 1773. 39. ELDEPRA~I, M. I<;., ELD~THWI, A. T., .\ND O’BRIEN, K. D. (1971) Proc. .Vaf. Acad. Sci. USA 68, 1047. -4., M~sr:z, A., 40. BO~I~GEOIS, J.-P., I~TTKR, FHOMAGROT, P., B~QLJI’T, P., ASD CH.\KGI.:UX, J.-P. (1972) Fed. Eur. Riochew Sot. Letters 25, 127.
373
AcCh-RECEPTOR
41. KARLIN,
A.,
PRIVES,
J.,
DF,AL,
W.,
AND
WINNIK, M. (1971) J. Mol. Biol. 61, 175. 42. LEVITZKI, A., AND KOSHLAND, D. E., JR. (1969) Proc. Mat. Acad. Sci. USA 62, 1121. 43. KATZ, B., AND THFSLEFF, S. (1957) J. Physiol. 138,63. 44. BENNETT, M. V. L., WURZEL, M., AND GIXJNDFEST, H. (1961) J. Gen. Physiol. 44, 757. 45. KARLIN, A. (1967) J. Theor. Biol. 16, 306. 46. CONWAY, -4., AND KOSHLAND, D. E., JR. (1968) Biochemistry ‘7, 4011. 47. RAFTERY, M. A., SCHMIDT, J., AND CLARK, D. G. (1972) Arch,. Biochem. Biophys. 162,852. 48. ELDI:FR.A\I’I, M. E., AND O’BRIJGN, K. D. (1971)
Proc. Sat. Acad. Sci. USA 68, 2006. 49. ELD?;FRA~I,
hf. E., ELDEFRAWI,
A. T., AND NeuroDekker, New
O’BRIEX, 1~. D. (1973) in Methods them. York.
(Fried, li., ed.), Marcel (in press).
50. WI:BB, G. 1). (1965) Hiochim. Biophys. Acta 102, 172. 51. LEUZINGER, W., ANI) BAKER, A. L. (1967)’
hoc. Snf. Acad. Sci. [ISA 7, 446. 52. DUUAI, Y., SILM.\N, I., KALDXRON, N., AND BL~XB~I~G, 8. (1972) Riochim. Biophys. Ada 268, 138. T. L., CHANG, H. W., AND CHKN, 53. ROSENBERRY, Y. T. (1972) J. Biol. Chem. 247, 1555.
OP
BIOCHliiMISTIZY
Purification
and
AND
BIOPHYSICS
Molecular from
Properties Torpedo
M. E. ELDEFRAWI Section of Neurobiology
3@2-373 (1973)
169,
and Behavior,
of the
Receptor
Electroplax A. T. ELDEFRAWI
AND
Cornell
Received
Acetylcholine
May
L~niuersity,
lthaca,
Xew York 14860
17, 1973
The acetylcholine receptor of Torpedo electroplax is purified by aflinity adsorption using cobra toxin (,Vaja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 rnv benzoquinonium produces a protein that binds a-[1251]bungarotoxin but not [3H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 M carbamylcholine produces an acetylcholine receptor protein that binds [3H]acetylcholine, [“Hldecamethonium, [3H]nicotine, [‘“C]dimet)hyl-d-tubocurarine, and cY-[‘2SI]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine recept)or (as measured by acetylcholine binding) averaging 69.2 Z+Z14.6yc. The purity of t’he isolated acetylcholine receptor protein is estimated to be at best 87% , as judged by disc gel elect,rophoresis and electrofocusing. The purified acetylcholine receptor binds 7.X nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K, = 0.02 PM) and 5.1 nmoles a lower a&nit3 (KD = 1.97 PM). If the protein concentrat,ion used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations 77p to 10 PM. Binding of acetylor robra toxin and was partially choline was totally inhibited by a-bungarotoxin blocked by four nirotinic drugs, but, not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.
Isolation of acetylcholine (AcCh)’ rcceptors from electric organs of fish has advanced from the stage of identification of the correct macromolecule in subcellular preparations, ut’iliaing binding of reversible ligands or neurotoxins, to that of different degrees of purification (1-3). We have previously identified AcCh rrcept,or molecules in membrane preparations from the electric organ of the cl&ric ray, Torpedo marmorata, by virtue of the characteristics of its AcCh binding, which was reversible, of high affinity and blocked only by nicotinic ligands (4, 5). It 1 Abbreviation: a-bungarotoxin.
A&h,
acetylcholine;
cu-BGT,
also bound nicotirw, dwamethonium, and curare (6) and its concentration was equal t’o that reported for the wbungarotoxin (a-BGT) binding molcculcs in the samr tissue (7). We have also shown t,hat these recept,or molecules could be solubilizcd by various detergent,s and retain their drug specificity (8). Conventional biochemical techniques, e.g., gel chromatography, elect’rophoresis, electrofocusing, or den&y gradient centrifugation, have been camployed, wit’h limited success, in the purification of AcCh receptor (7-10). Their drawbacks were’ cithrr bhe low level of purification achieved or the 362
Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
PlU)PEltTIES
OF PUIZIFIEI)
denaturation of t’hc receptor. The highest degree of purity of a functional receptor reported was 6.3-fold, obtained by electrofocusing (10). The recent introduction of the technique of afinity chromatography (1 l13) had a gwat. impact on the progrckss of rwcptor purificat,ion. The method is bawd on the ability of protcGw to bind certain ligands rcverslbly and with high affinities, so that Ohe highcr t,h(l nffinit~y the desired protein has for the ligand that is coval~~ntl~linked to t,he solid support,, tho more prc,tt>in will bind strongly and the more nonspwifitally bound proteins \vill desorh by elution with buffers. Thr A&h rccept,or from clectric organs of Totpedo and the clrctric ccl, IClectrophorus electricus, haw bwn purificid to varying dcgrces by affinit,y Columbus (16 lS), but, thwc is no report yet, of the>isolation of a totally pure: AcCh receptor. The Sepharow-linked ligands uwd ww either quat’carnary ammonium compounds (lZ, 16, 18) or the specific nwrotoxin from cobra (14, 17). Wc have used a phenyl t.rimethylammonium ligand (19) but the purification of AcCh recaptor from Tel-pedo \vas only lo-fold and t,hc recovery was very Iow (Eldcfrawi, unpublished). In the present cbmmunication, we describe an affinity m&hod well adapted for large scale production of a highly purified AcCh receptor from Torpedo rlcctroplax. This receptor protein is identified by virtue of the characteristics of it,s binding of its t,ransmitter, AcCh as well as other rcwrsihltt ligands, and cu-XT. Also, thr prop&w of the purifictd A&h rowptor are compared to thaw of its mr~mbranc-bound or solubilizcd countwpart, and its amino acids arc analyzed. MATERIALS
AN11 METHOl)S
Mnterials. Source of the AcCh recept,or is the lyophilized pellet of 12,000y of l’orpedo n~~rmora~a electroplax, stored under nitrogen at -20°C (20). [3H]Acetylcholine (48 mCi/mmole) was obtained from New England Nuclear, and [‘%]dimethyl-dtubocurarine (119 mCi/mmole), [3H]nicot.ine (1.7 and ~Hjdecatnethonittm (67 mCi,’ Ci/mmole), mmole) from Amersham Searle. Their raditrchemical purity was periodically checked by chroof the krait Bu7/garu,T matography. Venoms lnullicinclus and the Thailand cobra Suj-n naje
A&h-RECEPTOR
363
.sia7,ce,,sis were purchased from the Miami Serpentarium. PuriJica~ion ad use of [he neurotositrs. a-Bungarotoxin was isolat,ed in pure form by electrofocusing followed by gel filtration on Sephadex G50, and its idetrt.ity checked by its blockade of [“H]AcCh binding by the l’orpedo AcCh receptor and by autoradiography on mouse diaphragms (21). It was iodinated with [12511 by Sepharosebound lactoperoxidase, as described for other proteins (22), to give 0.X Ci,‘mmole. The ncurotoxin from the cobra was purified, then covalently fixed to Sepharose -IS following the method of Karlsson el cri. (14). The concentration of toxin used in this reaction was 0.25 mg toxin per ml of packed gel. The affinity gel was stored at 4°C in the presence of 10-j M hierthiolate and t~setl within a mont,h. Isolatiot~ of A&h rereplor b!/ u.@r~if!l udsorp/io,/. For eacah run, 1.68 g of the lyophilized pellet of 12,OOOg (from 120 g of ?‘orperlo electroplax) was homogenized in 60 ml of a modified Krebs original Itinger phosphate buffer (23, 24) containing l(A Trit,on X-100. This fluid fraction was then shaken for 30 mitt at. 4°C and centrifuged at, 105,OOOgfor I hr. Fifty milliliters of the supertrata.nt. was added t,o 25 tnl of packed aflinity gel, which had been washed with 100 ml of the Krebs-Ringer buffer, and t,he mixture was stirred for 1 hr in a loo-ml beaker. It was then filt,ered on a glass filter and the gel, with the A&h receptor adsorbed, was l.ransferred back t,o the beaker. Fifty milliliters of Ringer with only 0.17; Trit.on was added, and the tnixt~ure stirred for 20 mitt, then filtered on the glass filter so as t,o remove light,ly adsorbed or nonadsorbed proteins. This step was repeated once more. Then 50 ml of 1 .M SaCl in Ringer containing U.Iyo Triton was added to the gel and stirred for 20 min before filtration, to remove nonspecifically bound proteins. This step was also repeated. To the gel, 25 ml of 1 M carbatnylcholine (Aldrich Chemical Co.) in Krebs-Ringer brtffer (withottt any Triton) was added, and the mixture stirred for 16 hr. Although the A&h receptor has a lower affinity for carbamylcholine than the cobra toxin, the high concentration (1 M) of carbatnylcholine is sufficaient to desorb A&h receptor from Ihe toxin which is covalently bound to the gel. All steps were carried out) at. room temperature. To collect AcCh receptor in the carbamylcholine solution, the mixture was filtered on a Millipore filtration assembly using 0.35 pm HAWP filt.er to eliminate any bacterial rontamination. The filtrat‘e was loaded int,o dialysis sacs and dialyzed extensively in several changes of 5 mx dibasic sodium phosphate (pH 7.4), until the concentration of carbamylcholine dropped below lO-‘O M. Protein was assayed by the Lowry method
364
15Ll~l5F1~AWI
ANl)
I51,l~I~:FI:AWl
after whirh the gels were stained for 2 -3 hr with (25), using as a standard crystalline bovine serum 0.2jf’; Coomassie brilliant blrlc and dcstainc~tl albumin from Sigma washed with ethanol and with 7(‘;, acetic* arid for several hol,rs. The gcll dialyzed against Tris-acetate-phosphate brlffer, was scanned at 570 nm in a l~e(~km:tn Acta I I I pH 7. Activity of AcCh esterase was assayed hy spec~trophotornctcr. the Ellman method (26) and carbohydrat,es by the ilnthrone procedure (27). I: I~:S[JIi TS, Rir,di,/g of cho/i//ergic liga,lrls. Binding of the reversible ligands was measured by equilibrium Vvo shall rwtrkt the uw of the term dialysis as previously descsribcd (5, 2X). In the (aase A&h rcwptor, throughout prowntation of of [aHlArCh, Tetrnm was added to the pllrified th(k Itwults, to the molccul~~sthat bind A&h receptor preparation and dialysis medirlm at 10 and show th(l prqwr pharmacological spcciand 1 ~1w, respectively, so as to totally inhibit fkity, and w shall quantit!atc: it according the minute amount, of AcCh esterase present (4). to tho numbw of A&h binding sites. Trypsin (EC 3.4.1.4) (Type 1) and a-chymotrppI’urz”licatior~ oj A c(lh receptor. The capacit) sin (EC 3.3.3.5) (Type l), both from bovine g(,l to wniovc: pancreas, are from Sigma. They were used at 1 of thr cobra t,oxin afiinity mg,‘O.2 mg of pllrified AcCh receptor in 1 ml, and A&h rcwptor from the: extra& of Torpedo incubation was for 1 hr at 23°C. Binding of ~1~ ckctroplax in Iiwbs original Ringer plus 1 ‘2 1’“5I]BGT was assayed by gel filtration on an X-in. Triton was dctcwnincd bv incubating etch Sephadex (+-SO rolumn with a void volume of 3.5 1 ml of p&cd gcll n-it,11dkfwcnt volun~cs of ml. The A&h receptor-a-B(;T complex elrrted in t,lw Triton clxt’ract ; thq aftw 1-hr incubathe void vollune, whereas fret a-B($T, render the tion, the affinity gc~l \vas romowd by filtraexact assay caondit,ions, eluted in 7-8 ml. Onetion and [:3H]AcCh binding to the filtrate hundred-nucroliter samples were t,aken from cac.h d(~tcwninc~d. FV:(bfound that 1 ml of paclwd fraction, and the radioactivity counted. gel cwuld adsorb 2.3 nmol~s of AcCh binding Amino acid c~~ral?/sis. The purified A&h recoupsitw u+cn it ~vas incubattrd with 5 ml of th(a acid at t,or was hydrolyzed in 6 N hydrochloric 110°C ill evacllated sealed tubes for 20 or 40 hr, Trit,on clxt,ract (Table 1). WP wlectrd the and subjected to amino acid analysis in a Beckman ratio of 2 ml of Triton oxtractS (-1mg protcki/ model 120C analyzer accaording to the method of ml) to 1 ml of pwlcc~d gc,l, so that, all A&h Moore and Stein (29). Analysis for cysteine was rcccptor prcwtwt, would adsorb to the gcsl. after subjection to performic acid oxidation a(:Ilwovwicw of protclin, A&h wt(wsc, and cording to the method of Hirs (30). Tryptophan iIcCh rwytor arc prcwntcd in Table II. was assayed spect,rophotometricallJT (31) and also, Bawd on pmoh~s AcG bound/mg protctin, by chromatography following p-toluenesrllfonic, A&h rcwptor uxs purified by this prowacid hydrolysis (32). To estimate the concentradurc: 25fold. In 13 runs, rwwwrks of an tion of Triton X-100 present in the prlrified AcCh A&h rCMq)tcJr actiw (i.c>., A&h-binding) recept,or, we compared its uv absorption at, 221 and 280 nm with a st,andard (nl1rve made with a constant conc*entration of bovine serum albllmin (0.2 mg/ml) and concent,rat ions of Triton varying from 0.005 to O.l“i;. EZrclrofoc,/csi,cg. The LKB Produkter column (110 ml rapacity) was used as previously described W-9. ‘,g; of AcCh “moles of nmoles of ml of extract A&h receptor AcCh Gel clec(rophoresis. The met,hod used was that (4 mg receptor adsorbed& receptor l)rotein/ml) of Davis (33), but only running gel was used, so remaining add& added to that there was no previous c.oncentration in a after 1 ml of spacer gel. One-hundred microliters of purified incuhationrL packed gel AcCh rereptor fraction containing 15 pg protein was diluted with an equal volume of 40% sucrose 0.664 0 100 1 blue added. and a drop of 0.059; bromophenol 100 1.328 0 2 This mixture was layered on top of a 7’;io acryl0.M !I7 1 ,992 3 amide-0.18’; bisacrylamide gel (pH X.9). A Buch2 Cifi 18 9.i 4 ler analytical disc gel elect.rophoresis was used, 50 8.1, 3 ,320 ,i __. and constant current of 1 mA/tube was applied at. tL AcCh receptor is herein llsed t,o mean the first,, then raised to 3 milltube aft,er the marking [“H]AcCh binding sites. dye had penetrated the gel. The rtln lasted 2 hr,
PI’,OPElLTIISS
OF PURIFIEL, TABLE
A&h-CECGPTOlt
II
Ii M;~VISKIKS OF PROTHIN, ACCH ESTIXLWC .IND A&H E:XIW~IMI~:ST ny AFFINITY ADSOIWTI~N Fraction
Protein me/ fraction
l’g
Triton in Ringer extract (50 ml) l’j;, Triton in Ringer extract after adsorption First, O.l$;, Triton in Ringer Second O.lP’; Triton in llinger First O.lc/;, Triton, 1 M ISaC in Ringer Second O.lcz Triton, 1 1% NaCl in Ringer A&h receptor fraction desorbed with 1 JI carImmylcholine in Ringer Total “; recovery .4verage rewveries experiments
of
15
I~IXICPTOR ORTMNICD IN ii PURIFIC.ITIOS TO THR NI,:UXOTOXIX GEL AcCh esterase
C” (’
365
A.Xa,n/ fraction
C’ ic
i\cCh receptor pmoles AcCh hound/mg protein at 1 Pi AcCh
c-c
140
100
x00
100
206
100
04
66
550
69
0
0
33 5.4
23.7 3.x
16X 54
21 6.7
0 0
0 0
2.2
1 .6
14.5
1.x
0
0
0.8
0.5i
3 .5
0.44
0
0
1.1
2.x
0.1
0.001
9x.5 100.45 Ik6.14
rangod hct~wcw 55 and X2 76, with an awrage of 69.%%. Thc~ affinity gcal I\-as recycled hi washing with 200 ml Ringer and rwwd, but rwovcrir~s of A&h rcwptor awraged only 40 %, \\hich might; indicatcb that a few AcCh rcwptor mol~culw rcmaiwd attnchcld to the gd. I)eso,ption by benxoyuinoniuin compared to ~at.banz~lcholirle. A Triton cbxtracstof Torpedo
5135
!)8.9 101.08 rtll.78 _____
72.5
72.5 60.2 fl4.6
not bind [3H]AcCh. To determine whether thcb abwnw of binding of cholincrgic drugs b>~ the hc~IIzo(~uinonium-dt~sorhrd fraction \vas dw to df>naturation of the rwq~tor, WC incubat8ed 1 5’; Triton extract of Torpedo c4cctroplax \vit’h 10 rn>r henzoyuinonium for 16 hr, thcln dialyzed out this ligand. X&h binding \vas decreased by 80 %. 1lLolecular properties
of the AcCtl
receptor.
c4wtroplax was add(bd to t8hc affinit’y gcbland Binding of [“H]AcCh to the purified X&h twatcbd as dcscribt~d ahow, but in t’hc final rcwptor ~vas mclasurcld ovrr the concentrastop, dworption was awwllplishcd by 10 rnx tion range’ from 5 no t’o 11 q. The Scatchard hc~rlzocluinonillrll insttbad of 1 RI carbamylplot is prcwntod in Fig. 2a, and demonstrates choline:. Aftw wduction of hcnzoquiIlonium the prwcnw of two afini&s (possibly reprcto a conwntration below 1 pnr by dialysis, scnting t)wo sites). Iterative analysis of the thcb fraction bound w[“~I]BGT as did th(x data by II311 360 computw givcu a high car~)am~lcholinct-dcworb(~d fraction (Fig. 1)) affinity component with a dissociation conbut th(b hcnzoquinonium frakon (having stant (Ku) of 0.0% I.~I and a maximum (‘onprotein qua1 to X0 ‘2 of that of thcx car- wntration ([B],,,,,) of 2.7 nmol~~s mg probamylcholinc: fraction) did not, bind AcCh, tAn, and a lo\\- affinity component with a ~iiwtirw, d(carnothoniurn, or dimc+hyl-rlK, of 1.97 PM and [B],,, of 5.1 mnolcs/mg tubocurarincb (Tabk III). III anoth(lr VX- protAn. Th(w numbers add to giw a total pckmc~nt,, aftc,r dworption by b~wzoquinonX&h-binding sitw of 7.8 nrnolcs smg proium, twat,mcbnt of the go1with 1 11carhamylt8c>in. Thcwc~ valuw arc’ based on protein choline as prwiously dwcribcd rwovcwd analysis by th(l Lowry method (25). When an additional 15 %, of thr protck, \vhich did protcin \v:ts wtimated from amino acid
360
ELDI’FRAWI
ANI)
f~:LDI’FRAWI TAL-11,IS I I I
lot
BINDING OF CHOLINEKGI(' I,I(:.\NDS I%?' Two I
Cholinergic
ligand
Binding by two preparations (pmoles bound/mg protein) “aihb$l$ desorbed
[3H]AcCh (1 /XVI) [14C]I>imethyI-d-tubocurarine (0.09 PM) [3H]Nicotine (1.4 PM) [3H]l)ecamethonium (I .4 PM) u-[‘251]Bungarotoxin
TABLR
Benzoquinonium desorbed
.i 13.‘, 425
0 0
1016 2370
0 0
2515
2.580
IV
BI,OCKM)E Froct,o” number
OF [“H]AcCH BINDING (arr 1 PM) TO PGRIFIED A&H RECEPTOR ny VARIOUS
CHOIJNEIWIC
FIG. 1. Gel filtration on Sephadex (i-50 of different fractions after incubation with ,-[1251]BGT. (.......) a-[1251]BGT alone; (A-A) original Triton ext,ract of Torpedo electroplax; carbamylrholine-desorbed fraction; (O-----O) (m - -m) benzoquinonium-desorbed fract,ion. Flow rate is 25 mI/hr, fraction volume is 1 ml.
analysis, its concentration in t’hc purified AcCh receptor fraction was 25 %I lower. Based on this lower prot’ein value, tho total specific [3H]AcCh binding in the purified AcCh receptor is calculated to bc 10.4 nmolcs/mg protein. The Hill plot of the data (Fig. 2b) gives a lincl wit’h a slope of 0.5 and the R, value (see Ref. 34) is calculated to be about 500. Incubation of the purified AcCh receptor with trypsin or ol-chymotrypsin for 1 hr at 23°C reduced the amount of [3H]AcCh bound at, 0.1 PM by 34.7 % and 66.5 %, rcspcctively. When binding of [3H]AcCh at 1 PM by the purified AcCh rwcptor \vas measured in t’hc presence of cholinergic drugs (100 PM) in t’he dialysis bath, the nicotinic OIWS blocked the binding from 42 to 77 %, but th(> muscarinic ones did not (Table IV). Addition of (Y-BGT or cobra ncut8rotoxin to the purified &4cCl1receptor rcsultod in t,otal blockade of Arch binding.
I)RUGS
.\ND
Cholinergic ligan& _____~~
NEUROTOXINS
% Blockade
Nicotine 77 d-Tubocurarinc 65 1)ecamethonium 43 Benxoquinonium 42 Pilocarpine 0 At,ropine 0 Cobra neurotosin 100 amBungarotosin 100 .~~ ~~~~~ ~.__ ~____. 11Each drllg was added to the dialysis bath at 100 PM, but each neurotoxin was added to the purified AcCh recept,or to give a final concent,rat,ion of 1 PM, then equilibrium dialysis was started 30 min later.
CM clcctrophorwis of the purified fract’ion, in the absence of SDS, showed that the protein did not precipitate and that 87% of it migrated as one band a little into the running gel (Fig. 3), which indicates a molecular weight abova 300,000. When a-[‘2”I]BGT was added to tho purified AcCh rwqtor and then rlrct’rophoresr:d without SDS, the radioactivity was rcst’rictcd t’o t’hat major band. Electrofocusing of the purified AcCh receptor shoLvc:d that -93 % of the protein had an isoelcct,ric point around 4.8 (Fig. 4). The purified AcCh rcwptor showed rcmarkable stability as judgc>d by its binding
PROPERTIES
OF PURIFIED
of [3H]AcCh. When stored at 4”C, it lost 10% of its binding after 14 days, but when stored at O”C, them was no loss up to thrett weeks. The concentration of Triton in the purified AcCh receptor fraction n-as calculatcd to be about 0.007 %,.
A&h-RECEPTOR
The amino acid analysis of the purified AcCh receptor is present,ed in Table V. The high concentra6ion of acidic amino acids reflects the acidic nature of the AcCh receptor. We found equal amounts of carbohydrates in the AcCh receptor preparation a.s
1I
-3
-2
367
-1
0
Log [Ll
FIG. 2. Binding of [‘H]AcCh hy the purified AcCh receptor. (a) Scakhard plot B. hmount, bound in pmoles/mg protein; I, = [“H]acet.ylcholine concent,ration in FM. (b) Hill plot of the data. B’ = [B]/[B],,,,, where [Blmax is obtained from the computer analysis of the Scatchard plot. [L] = [“H]AcCh concentrat,ion in PM.
36s
El
)EFltAWT
AN11 ELl)EFKAWI
in blanks of Ringer or bovine serum albumin processed through the affinity adsorption procedure. Thus, it, seems that the source of carbohydrates is the Sepharosc used in affinity adsorption, which is a polymerized dextran.
3. Scan of a gel st,ained with Coomassie FIG. Brilliant Blue after electrophoresis of the purified AcCh receptor.
FIG. 4. Electrefocusing cal density of the fractions at low pH (<3).
Identification of A&h receptor. Binding of (Y-BGT has become a favorite index for t,ht: identification of nicotinic A&h receptor (3, 7, 3.5, 36). Yet there arc: many observations that prow: t#he nonspecificity of cr-BGT, especially when used at high concentrations or in homogenates. We found that (u-BGT bound to membrane preparations from the axons of lobster walking legs, where no AcCh rcwptor is known to exist (37). Thwt also macromolcculcs cholinc~rgic-binding bound A&h and nicotine, but whereas AcCh differentiated br~twcctn thcsc axonic mwromolecules and tht: AcCh rccept,or in having a much lower affinity for tho former, nicotine and CV-BGT did not (37). It has also bwn reported that at high concc,ntrations, cu-BGT &a~bound to tho sarcoplasm of 111ous~~ phragm (36). These data, plus the present, finding that, b(lrizocluinoniulri possibl!- dcnat,uwd AcCh IYYx!~~t(Jr and dworbed :I protein that bound o(-BGT and not ,4&h or othw reversible cholinergio ligands (Table III), indicate that total dopcndcnw on binding of o(-BGT for idt4ficatitm of thca purified A&h receptor or calculat~ion of its dcgrec: of purity (15, 17, 18) is quite inade-
of purified AcCh recept,or using ampholyt,es (pH 3-10). Optiis measured at 2X0 nm after correction for the high absorption
quat(‘. WC, thtwforc~, still strew that. the binding is pharmacologically specific (5, S, 10). Aftor all, binding of insulin has bwn host index for AcCh rcwptor is its rwcrsibl(~ binding of its trwnsmittw X&h, under COIN- omploycd to identify the insulin rcwptor ditions and at concc~ntraticJns \vhwc: thus (11) and noradrenaline the p-adrcncrgic rewptor (3). T.4BLK
Lysine Histidine Arginine Aspart ic acid Threonine Serine (illIt amic acid Proline Glycine Alanine Half cyst,ine Valin? Met hionine Isoleucine Leucine Tgrosine I’henylalanine Tryptophan Ilexosamine
1
6.1 2.1 3 .i 11.x 6. :3 7.1 10.5 (i .2 Ii .4 6 0
4 .:I 2 3 .i .4 10.8 4 .:3 ti .!I 9 .4 8.1 7.7
2 .o .i .i
1.1 7 .o 3.0 :<.7 9.0 3.8 .i .:3 2.0 1 6
1.7 5.2 0 .3 3 6 4.4 2.111 06
.i ,i
A.fitlity adsorpiiott atrcl degree of purz’jication. The> abovo procedure of affinity ad-
4.8 2.1 .i 0
12.6 4.1 6.8 11.1 i 8.8 i.4 0.0 fi 0 1.3 4 .o 8.2 2.9 .5 1 -
4.G 2.3 .-I 2 13.1 4..i 6.8 10.4 .i .!J x.7 (i .2 1 .fi
7.1 2.i 3 .8 8.6 3 (i .i 3 2.0
1 :%
‘1 These values are calmdated from data on tolrlene-extracted AcCh esterase from E(ec/rophuUIS electroplax presented as pmoles per sample or the integers of amino acid residrles assuming four histidine residues per minimum molecular weight (;il), and t,hey are the average of a 24. and 2“-hr hJ-drolysis of the prot,ein. ‘1I;ach number is an average obtained from three different preparations from Ei;‘ler:lrophorras electroplax (.i2). The AcCh est,erase was either homogenized and t rypsin toluene-extracted, digested, or subjected to both treatments, followed by af6nit.y chromatography ’ These values are calculated from average data on 3 samples presented as residues per 12 valinc residues. Acetylcholine esterase 11sed was tolllene-extracted from Elw/rophorus electroplax and purified by afhnit y chromatography (.X3). ,’ This value was determined by two met hods (81, 32). r We found 1 mole of carbohydrates per 10,000 daIt,ons of prot,ein. An equal concentration was found in a blank (I$; Triton in Uinger) and in a bovine serum albumin preparat,ion, both srlhjected to the same affinity adsorption prorcdllre.
sorption apparclntly offers scwral ndvantagcis owr the USCof affinity cwlumns. First is tlic, fact that it is not limited to the size or rat(> of flow of a colunm. WV haw purificbd Z-10 mg of AcCh rcwptor in a single run, and it is possible to process up to 1 g of A&h rcwptor. The awragc rccowry value of 69.2 “;) (T&1(, II) is the highest yet rcportcd, eompawd to 30-50 (l:‘,), 55 (18), and 60 !‘; (l(i), valuw bawd on LU-BGT binding. WV haw purified the XcCh rcccptor and cLnd(bdI\-ith 7.S to 10.4 nmol~~sof AcCh-binding sitca pw mg prot’cin, as judgc>d by sprxrific A&h binding (Fig. 2, Tabk IV), which arhiww a %-fold purification by afFinit> adsorption. Thrsc valurs may bc compawd with 2.7 and 6 nmolrs cu-BGT-binding sitwl mg protck from electric organs of narcintl and Torpedo califortrica2, rrspcctiwly, quivalcnt to 13.9 and 2%fold purification. Thcscl results \vcr(’ obtained by Schmidt and Raftclry using affinity chromatography on cowtlcwtly Ii&cd quaternary ammonium ligands ;t~l clution with NaCIkSa phosphate l)uff(br (16, IS). I
370
ELDEFRAWI
AND
2-3.3 nmoles/mg protein based on ol-BGT binding, and the multiple of purification by the affinity chromatography step was 130fold. The purification multiple that’ is theoretically feasible to obtain a pure AcCh receptor depends on t’he original concentrat,ion of AcCh receptor in the tissue. For example, we have shown by reversible binding of cholinergic ligands that the concentration of AcCh rcceptor in Electrophorus electroplax was 0.02-0.03 nmoles/g tissue (39), compared to a concentration of 1 nmoles/g of Torpedo electroplax (5, 20). Similar values mere obtained using a-BGT (7), cobra t,oxin (40), or maleimide compounds (41). Since the concentration of protein/gram tissue is similar in the two electric organs (20, 39), to arrive at an equally pure AcCh receptor requires 2030-fold more purification from the eel than does that from Torpedo. The purification that we have presently achieved is 2.5fold by affinity adsorption, Z-fold through membrane preparatjion (20), and 2-fold through Triton solubilization (S), adding to a total of 100-fold purification. Based on prot’ein and ol-BGT distribution in elcctrophoresis (Fig. 3) and the electrofocusing data (Fig. 4), it seems that the AcCh receptor obtained is, at best, 87% pure. This is still higher than everything so far reported on AcCh receptor of Torpedo californica or T. marmorata, as evidenced by the multiplicity of prot,ein bands in clectrophoresis or electrofocusing, respectively (18, 14). Total purificat,ion of AcCh receptor of Torpedo may thus require a II&fold lcvcl; accordingly, tot’al purification of AcCh receptor of Electrophorus electroplax would require a tot’al of 2300-3450-fold. Thcrrfore, the tot’al degree of purification so far achieved for Electrophorus AcCh recrptor, given at 1300-fold with a-BGT binding of 2-3.3 nmolcs mg protein (15), wprcsents only 40-60 % purification. Properties of the puri$ed AcCh receptor. This purified AcCh receptor still binds [3H]AcCh with two high afkitiw (Fig. 2a) as it did when at’tached to membranes (5) or after solubilization (8). The ratio of the low to high affinities is changed from 1.9 to 2.25 t’o S.3 in the purified, Lubrol-solubilizcd, and membrane-bound AcCh receptor, respec-
ELDEFRAWI
tively. Similarly, the Hill wefhcient is changed from 0.98 (5) to 0.66 (S) to 0.5 (Fig. 2b) in the three preparations, rospcctively. A Hill coefficient of 0.5 and an R,qof 500 for the purified AcCh receptor arc indications of ncgat’ivc: cooperativit!. (34, 42). Desensitization has been obwrvcd in electrophysiological expclriments in musclw and Torpedo cblectroplax (43, 44), whrrc exwss agonist conwntration kd to a reduct’ion in response, a phwomcwon which may bc considered as a case of ncgativc cooperat’ivity. Positive coopwativity has also bwn obwrvcd in a monoccWar preparation of WI c,lwtr+ plax, whore a Hill rorffick!~t~ of closc~to 2 \vas obtained with carbamvlcholitw (1.5). Such physiological responses arc tht> end result of a number of molecular cwnts t,hat, start’ with binding of a ligand to tlw A&h rcwpt.or. Therefore, tho clectrophysiological observations might, bo refkct~ions of events at the ligand binding step or subseqwnt OIIW. Our present observations suggest that, ncgat)ivc coopcra0ivity m:ty occur at th(> binding step. Although WChave not obscrvcd positive cooperativity in thr binding of AcCh to the purified A&h rweptor, it is possible that it may bc detected if binding is studied at low conccntrat’ions of [“H]AcCh which increaw at very small incremc:nt’s3. A single protein such as AcCh receptor may well clxhibit both positiw and ncgativc coopcrativity, depending upon the part’ of the ligand concentration curve under cxaminat’ion (46). When cu-BGT binding was studkd to ~lwkoplax of T. califorrzica, what may bcl t\vo sites was also found (47). Kot only do th(h conc&rat,ions of binding sit,w differ in thch purified AcCh rwxptor, but thcbaffinitks also decrease about IO-fold owr thca Lubrol-solubilizcd AcCh rcwpt~or. Anoth(ar change is the absence of aut,oinhihition up to 11 ~LJTA&h, t,hough n-c’ had ohswwd it abow 1 pc1-\1 in the mrmbranc prcbparat’ion (4X) and also th(> solubilizcd preparation, by c~quilibrium dialysis or by ultrafiltration (49). This phonomrnon was not, dw to wnctIivation of AcCh 3 After submitting this rnanusrript, we det,ected positive cooperativit,y it) rlrCh billding to this purified AcCh recept,or preparation at, i\cCh COIIcentrat,ions ranging from 0.0007 t,o 0.01 &l (Eldefrawi, M. E., and Eldefrnwi, A. T., Rioche/w. Pharntacol. Rep. Cotuv~. 1 (in press)).
PROPERTIES
OF PURIFIED
&erase, because we did not detect any increase in the concentration of acet’ate after dialysis at high concentrations of AcCh (Eldefrawi, unpublished). It, may be Ohat a polypeptide or a phospholipid responsible for t,hc aut’oinhibition was lost during purificaDion, or it is possible that the phenomenon may still occur hut’ at higher AcCh concentrations,. which \vould make it undet)ectable by cqmhbrium dialysis. On the other hand, the receptor protein is st.ill highly acidic wit,h an isorlcct,ric point of 4.S, which is not significantly different from t,hat of 4.5 f 0.2 we reportcld for the solubilizcd AcCh receptor (10). An interesting obscrvat#ion is t’he difference in binding of cholinergic ligands by the protein dcsorbed by carbamylcholine as compared to benzoquinonium (Fig. 1, Table III). The lack of binding of reversible ligands by t#h(abrnzoquinonium-dcsorbed protein may be due to a denat’uration possibly caused by an impurity present in the bcnzoquinonium solution or by the long exposure to the high concentration (10 m>r) of benzoquinonium used. Another alternat’ive is the incomplete removal of bcnzoquinonium by t,he extensive dialysis, which seems to bc a rem& possibility in view of the reversible nature of the in situ effect of bcnzoquinonium (50). To explain binding of LY-BGT in the presence of benzoquinonium, Tve would have to assume &her that a-BGT has separate binding sites from those of the reversible cholincrgic ligands or that the bulkier wBGT forms a larger number of bonds w&h AcCh receptor overlapping the areas of binding sites of the reversible cholinergic ligands. If one assumes that the Torpedo AcCh receptor is 57 % pure at the specific activity of 7.8 nmoles/mg protein (based on Lowry analysis) or 10.4 nmoles/mg protclin (based on amino acid analysis), then the molecular lvcight of t’hc subunit, which binds AcCh would be in the range of 83,000 to 112,000 daltons. Determination of the sizes of the subunit’s after different chemical trratmcnts, and following them by clcctrophorcsis, dynamic sedimentation, and electron microscopy, is under current investigation. Electron micrographs of the negatively stained purified AcCh receptor show doughnutshaped globular molecules (Salp&er and
A&h-RECEPTOR
371
Eldefrawi, unpublished). This highly acidic AcCh receptor protein has a high tendency to aggregate as we have previously reported (8, 20), which may indicate that there are areas of high densities of positive charge in the molecule despit’e its overall negativity. The degree of aggregation varies under different concentrations of salt or pH. The binding of [3H]AcCh indicates the presence of two affinit’ies (Fig. 2a), which may be due to two distinct sites on the molecule, two conformational states, or possibly different degrees of aggregation. The binding constants may change depending on the degree of aggregation and the detergent present, and thus may not reflect the in tivo situation. Many similarities were found between AcCh receptor and AcCh e&erase (e.g., presence in the same tissue fractions, similar concentrations, binding of AcCh, and comparable effects of chrmical reagents) that led to the earlier suggestions that the two are ident,ical macromolecules (see Ref. 1). However, diffcrmt purificat,ion methods have SUCceeded in partly separating these two macromolecules, and affinit’y chromatography was by far the best (14, 15, 18). In our purified AcCh receptor fraction, we calculate only one catalyt#ic site of A&h esterase still present’ for every 20,000 AcCh binding sites. There appears to bc more similarities than t,hcrc are differences in the amino acid content bct#ween AcCh receptor and A&h esterase (Table V). Thr basic amino acids make up approximately 12 mole % of either macromolecule, but AcCh rcccptor has about 33 % more lgsinc and 33 % less argininc than AcCh esterase. The acidic amino acids make up about 20-23 mole %, of either macromol~~culc, with approximately equal representation by aspartic and glut,amic acids. Acetylcholinc receptor has a higher molt % of t#hrclonine, half cystine, and isolcucine, and less of glycinc and valine. It stems that the source of carbohydrates in the purified AcCh receptor fraction is the Sepharosc used in affinity adsorption. It is still possible that a hexosaminc may be a component of the AcCh receptor molecule, but it,s presc’ncc might br: masked by t’he large amounts of carbohydrates in the fraction. By comparison, two of the analysrs of A&h esterase have reported the prrsrnce of hexosamine (51, 52). It would h(a more
372
ELDEFRAWI
.4X1) ELDEFRAWI
relevant to compare the two molecules from the same species, but unfortunately the amino acid analysis of Torpedo AcCh esterasc is not available. The reduction in [3H]AcCl~ binding observed after incubation of the purified AcCh receptor with trypsin or a-chymotrypsin poirks to the possible importance of the peptide bonds involving the carboxyl groups of arginine or lysine and tyrosine or phenylalanine for the activity of the AcCh receptor. The previously observed effect of 1,4-dithiothreitol in reducing AcCh binding by the membrane-bound AcCh recept#or (10) points to the importance of the disulfide bonds to AcCh receptor activity. These various bonds, and possibly others, are apparently essential for preserving the appropriate conformation of the AcCh receptor necessary for its binding of AcCh, and their relationship to the active site is yet to be elucidated. ACKNOWLEDGMENT We thank Mr. N. Balderrama for help in t,he electrophoresis experiments and Ms. S. Kane for technical assistance. We are grateful to Professor D. Wilson, Cornell University, for the time he spent in the analysis of amino acids and helpful discussions; and Professor T. C. Vannaman, Duke University Medical Center, for the analyses of tryptophan. We are also grateful to Professor R. D. O’Brien, Cornell University, for providing the lyophilized Torpedo electroplax used in the present research, which he collected at the Zoological Station, Naples, Italy; and the benzoquinonium sample presently used, which he received as a gift from Professor G. Webb, University of Vermont at Burlington. This research was partly financed by National Institute of Healt,h Grant No. NS-09144. REFERENCES 1. O’BRII~:N,
R.
D., ELDPFRA~I, M. E., SND A. T. (1972) Annu. Rev. Pharmacoz. 12, 19. 2. HALL, Z. W. (1972) Anuu. Rev. Biochem. 41, 925. 3. POTTER, L. T., AND MOLINOFF, P. B. (1972) i,l Perspectives in Neuropharmacology, (Snyder, S. H., ed.) p. 9, Oxford University Press, London. ELDF;FR~WI,
4. ELDEFRawI,
M.
E.,
BRlTT&:N,
A.
G.,
AND
O’BRIEN, R. D. (1971) Pestic. Biochem. Physiol. 1, 101. 5. ELDEFR~WI, M. E., BRITTIGN, A. G., *~.IND ELDRFR.ITVI, A. T. (1971) Sciejlce 1’73, 338.
6. ELDEFR.\\VI, MOUIt,
L.
M. P.,
I<., b:LDI:FR.\\\.I, .~ND O’BI~IKN,
A.
I:.
T.,
(;lI,-
1). (1971)
Mol. Pharmacol. 7, 420. 7. MIL~XJ, IL., MOJ~INOFF, P., .~ND POTT~;R, L. T. (1971) :Vaturc: (Lodo~) 229, 554. 8. ELJ)EFR.Z~I, M. E., I?LJ)I,:FK.\~vI, A. T., SXIFliJtT, S., .\ND ()‘BKII,:N, 1:. 1). (1972) Arch. Biochem. Biophys. 160, 210. 9. MEUSIXI~, J.-C., OLSEN, 1:. W., R/Ib:N,CZ, P., B
M.
E.,
EJ~DCFK.4w1,
A.
T.,
.\ND
PROPERTIES O’BRIEN, 104.
OF PURIFIED
R. D. (1971) Mol. Ph,armacol. 7,
29. MOORE, S., AND STEIN, W. H. (1954) J. Riol. Chem. 211, 893. 30. HITS, C. W. H. (1956) J. Biol. Chem. 219,611. 31. BENCZF,, W. L., >\ND SCHMID, K. (1957) A,la!. Chem. 29, 1193. 32. Lru, T.-Y. (1972) Methods Evzymol. 26, 44. 33. I)AVIS, B. 12. (1964) L~WL N.Y. .I&. ,%i. 121, 404. 34. KOSHLAND, I). E., JR., N~METHY, G., .z~u FILMF,IL, D. (1966) Biochebstry 6, 365. 35. STY-CKOWSKI, A, J., VOGEL, Z., AND NIREKHKRG, N. W. (1973) Proc. Sat. Acad. Sci.
USA 70, 270. 36. PORTER, C. W., CHIN, T. H., WIHXO~SKI, J., AND BARNARD, E. A. (1973) &V\‘atxre *Vew Riol. 241, 3. 37. DICNBURG, J. L., ELUXFRA~I, ?vl. E., AND O’BRIE:N, R. D. (1972) Proc. Sat. Acarl. Sci. USA 69, 177. 38. LEFKOWITZ, It. J., .\ND I%ABER, E. (1971) Proc. Nat. Acad. Sci. USA 68, 1773. 39. ELDEPRA~I, M. I<;., ELD~THWI, A. T., .\ND O’BRIEN, K. D. (1971) Proc. .Vaf. Acad. Sci. USA 68, 1047. -4., M~sr:z, A., 40. BO~I~GEOIS, J.-P., I~TTKR, FHOMAGROT, P., B~QLJI’T, P., ASD CH.\KGI.:UX, J.-P. (1972) Fed. Eur. Riochew Sot. Letters 25, 127.
373
AcCh-RECEPTOR
41. KARLIN,
A.,
PRIVES,
J.,
DF,AL,
W.,
AND
WINNIK, M. (1971) J. Mol. Biol. 61, 175. 42. LEVITZKI, A., AND KOSHLAND, D. E., JR. (1969) Proc. Mat. Acad. Sci. USA 62, 1121. 43. KATZ, B., AND THFSLEFF, S. (1957) J. Physiol. 138,63. 44. BENNETT, M. V. L., WURZEL, M., AND GIXJNDFEST, H. (1961) J. Gen. Physiol. 44, 757. 45. KARLIN, A. (1967) J. Theor. Biol. 16, 306. 46. CONWAY, -4., AND KOSHLAND, D. E., JR. (1968) Biochemistry ‘7, 4011. 47. RAFTERY, M. A., SCHMIDT, J., AND CLARK, D. G. (1972) Arch,. Biochem. Biophys. 162,852. 48. ELDI:FR.A\I’I, M. E., AND O’BRIJGN, K. D. (1971)
Proc. Sat. Acad. Sci. USA 68, 2006. 49. ELD?;FRA~I,
hf. E., ELDEFRAWI,
A. T., AND NeuroDekker, New
O’BRIEX, 1~. D. (1973) in Methods them. York.
(Fried, li., ed.), Marcel (in press).
50. WI:BB, G. 1). (1965) Hiochim. Biophys. Acta 102, 172. 51. LEUZINGER, W., ANI) BAKER, A. L. (1967)’
hoc. Snf. Acad. Sci. [ISA 7, 446. 52. DUUAI, Y., SILM.\N, I., KALDXRON, N., AND BL~XB~I~G, 8. (1972) Riochim. Biophys. Ada 268, 138. T. L., CHANG, H. W., AND CHKN, 53. ROSENBERRY, Y. T. (1972) J. Biol. Chem. 247, 1555.