Molecular structure of elongated forms of electric eel acetylcholinesterase

J. Mol. Biol. (1978) 125, 293-311

Molecular Structure of Elongated Forms of Electric Eel Acetylcholinesterase LILI AWGLISTER AND ISRAEL SILMAX Departmerd

qf Neurohiology

The Weizmann, Institute (Received

12 December 1977. rd

qf Science,

Rehovot, Israel

in rrvi.sed ,fovm I5 Ju,ne 1978)

Molecular forms of acetylcholinesterase extracted from fresh electric orgau t,issur of the electric eel are elongated st’ructures in which a multi-subunit Ilead is COIIelected t,o a fibrous tail. The principal form. 18 S acetylcliolinesterase, is of molecular weight approximately 1,050,000, contains about 12 catalytic subunit,n in its head, has a tail approximately 500 8, long, and aggregat,es reversibly at low ionic strength. Trypsin converts it to an 11 S globular tetramrr devoid of t-he tail and lacking the capacity to aggregate in low-salt solutions. Amino acid analysis shows that elongated forms of acetylc)tolinesterase significant amounts of hydroxyproline and hydroxplysinr. characteristic ponents of collagen. which are absent from 1 1 8 itc~tylc)lOlirlest,erasP.

contail) con-

Collagenase converts 18 8 acetplcholinesterase to a 20 S form whicli uo longer aggregat.es in low salt. Purified 20 8 acetylcholinesterase has about half the hydroxyproline and hydroxylysine contents of the 18 S enzyme, and physicochemical measurements indicate the formation of a, more symmetrical molecular structure without marked reduction in molecular weight. Sodium dodecyl sulfate/polyacrylamide pel elrctrophorcsis without reducing agent sI1ows that in 18 S acet’ylcholinesterase half the catalytic subunits arrh present as dimers linked by disulfide bonds. Tl~c remaining subunits migrate as larger molecular species which contain significant, amounts of llydroxylysinc, are specifically modified by collagenase and are corlvrrtrd t,o dimers and monomrrs hy trypsin. gel electrophoresis with reducing agent Sodium dodecyl sulfate/acrylamide t,wo polypeptides of molecular \veight,s reveals, iii 18 S acetylcholinesterase, 45,000 and 47,000 which are absent in the I1 S tetramer. They are readily digested by collapenase under conditions which do not affect the catalytic sllbIlnits, wit11 concomitant formation of a new 30.000 polypeptide. The above data can be rationalized by a model in which 18 S acetylctlolinest,crase contains three subunit tetramers, each linked by disulfidos to one strand of a collagen triple helix. Sodium dodecyl sulfate detaches those subunit dimars which are not covalently linked to the t,ail; trypsin attacks the distal portion of t,he collagen triple helix releasing discrrt)e tetramrrs, and collagenase specificall) attacks the triple helix near its midpoint, prodllcing a shortened st,rllct,ure irl whicll tjho residual tail still holds the tetramers together, but destroying the capacity for self-association at low ionic strengt’h. This latter property may be related to the postulated role of the tail in anchoring acetylcholinesterase to the fibrillar matrix of the basement membrane.

291

IA_ .\sGI,Is’l’I~;li

.\SI)

I.

Sll,hlAS

1. Introduction Aoet.vlcholinest,crase (acetylcholinc hpdrolaw. EC 3.1.1 .7) from &ctric organ t,issucb of the elect’ric eel, EZwtrophorus ~~P&~czI.Y. can Iw obt8ained in a numtwr of different molecular forms. Three forms can I)r tbxtra,ctcd from fresh tissue \j,ith sedirnent,at,ion coeficients of about, 18 8, 14 S and 9 S (Massouli6 & Rieger. 1969: Dudai et al.. 1972h). and all three have a unique elongated structure in which a mulbi-subunit head. containing the catalytic subunits. is connected to a fibrous tail (Dudai Pt al.. 1973: Rieger et al.. 1973). While the t’ail is of approximatel>r tht 1 same length in all t’hree forms. the electron microscope and physicochemical data suggest that the heads of the 9 S, 14 S and 18 S forms contain, respectively, one. two and three tetramers of catalytic subunits (Dudai et ul.. 1973: Bon it al., 1973.1976: Rieger et ~1.. 1973: Silman & Dudai, 1975). The principal molecular form extracted is 18 S AcChEase-I-. and the 9 S species is usually a minor comJ)onent. ,4utolysis and treatment with various proteases of electric organ tissue extracts lead t’o the appearance of additional molecula,r forms of AcChEase. Thus. both autolysis and tryptic digest)ion convert all three forms to an I1 S globular t)etramcr which is devoid of the tail. and. at the same time, the characteristic capacity of the elongated forms to aggregate reversibly at low ionic strength is lost (MassouliB et al.. 1970: Dudai et al., 1972a). Digestion of tissue extracts with collagenase produces another set of molecular forms as characterized k)y their sedimentation coefficient (Dudai & Silman. 1974c: Lwrbuga-Mukasa it al.. 1976: Rieger et ad.. 1976; .Johnson et al., 1977). Collagenase and proteases also detach nerve endings from the endplat’es of vertebrate neuromuscular preparations concomitantly with release of AcChEaw and digestion of t#he basement membrane (Hall & Kelly, 1971: Betz & Sakmann. 1971,1973). These observations. as well as the ready extractability of electric organ AcChEase by salt solutions (Silman & Karlin. 1967; Rosenberg it al.. 1977), raise thts possibility that in electric organ tissue. as well as in skelet’al muscle, AcChEase is attached t’o the basement membrane; since collagen is a major component of the basement membrane (Kefalides. 1973a) the tail of the enzyme might t’hus be a collagen-like structure whose role would be to anchor AcChEase to the basement membrane mat’rix (for a review. see Silman. 1976). This proposal is supported by the observation that purified elongated forms of AcChEase from electric organ tissue of both E. electricus and Torpedo cal
used:

AcC’hEasc,

Ltcrtylcholine~tara~H;

SIIS,

sodium

dodroyl

xulfate.

STRUCTURE

OF

.~C~TYLCHOLTNESTER.~SE

‘95

2. Materials and Methods Acetylcholine chloride, acetylthiocholine iodide, 1, IO-pher~ar~throline, dithiobis-(%Escherichia coli beef liver catalase, nitrobenzoic acid), phenylmethylsulfonyl fluoride, from Sigma; hactorial /3-galactosidase and bovine fraction I fibrinogen wcrc obtained collagenase (code CLSPA), trypsin (code TL) and soybean trypsin inhibitor (codt, STI ) from Worthington ; Sepharose CL-2B from Pharmacia, and decamethonium bromide front K and K. Iodoacetic acid and iodoacetamide were purchased from Fluka and rrcrpstallizc~tl from petrol ether and benzene, respectively, before use. [ 3H]diisoprop-lflllor~~pt Iospltut~~ Rabbit back skeletal muscle was purchased from tile Radiochemical Center, Amersham. myosin and bacteriophage T4 were gifts from Dr Raphael Lamed and Dr .Jacob Grimber~. respectively. Sucrose, buffers and salts were of analytical grad<%. Live electric eels, E. electricus, were obt.ained from M:orld\vide Scietltitio .luitnals. Ardsley, N.Y ., and killed by decapitatiorl irnrnrdiat,rl>before ,IW for prC*paratiotl (If AcChEase. (b) Acetylcholinesterase

preparations

Purified 14 S + 18 8 AcChEase and 11 H AcChEase were prepared by affinity cJtron:atography as described previously (Dudai et al., 1972a,h; Dudai & Silman, 1974a). Samples employed had specific activities of over 5500 units/o.n. unit of absorbancn at, 280 ,111,. where 1 unit is the amount of enzyme hydrolyzing 1 pmol of acetylctlolirl~/mitl ulld~r t Ire, assay conditions described before (Dudai et al., 1972a). 16 S + 20 S AcChEase, obtained by collapenasr dipestiorl of purified 14 S * IX S AcChEase, was repurified by adsorptiorl on ttlc same plietl~ltrimetll~-vlariilllorlilltllSepharose resin used for purification of 11 S AcChEase, ilr 0.1 nl-NaCl, 0.01 nl-sotlirlrn phosphate (pH 7.0), followed by elution wit,tl 10 mnr-drcern~tllotlium bromide itI t,lrr siuntb brdfer. 14 S and 18 S AcChEase were separated from each other by preparative sucrosfl gra,tlicbnt ccntrifugation as described previously (Dlldai & Silrnatl. 1974a), and 20 S and lfi S AoChEase were similarly separated. Extract,ion of AcChEase in the presence of sulfhydryl-blocking reagents WXH performed as follows: electric organ tissue (1 g) from freshly killed eels was homogenized in 2.5 ml of 1 &I-NaCl, 0.01 M-phosphate (pH 7*4), containing 1V4 ~-ptlen~lrnethyls~~lf~~~~~l fluorid
Collagenme

Protease-free collagenase was prepared from CLSPA-grade (Ilostrirli?cnh histolytictcwr collagenase (U’orthington Biochemicals) by gel filtration 011 Septkadex G200 according to Peterkofsky & Diegelmann (197 1). The purified collagenase was devoid of detectable caseinolytic activity by the Kunitz assay (Laskowski. 1955), i.e. it. contained proteolvt,ic activity corresponding to less than 0.1 o/o of that of an qua1 weight of trypsin. It \vas st,ored at -20°C in 5 mM-CaCl,, 0.05 ;M-Tris (pH 7.6). nntil rlsed. (d) Amino

acid analysis

Samples for amino acid analysis, each containing 0.4 to 1 mg protein, were tlydrolvzed and analyzed as described by Spackman et al. (1958). Hydroxyproline was determ”ined calorimetrically by a modification (Anglister et al.. 1976) of t,he procedure of Bondjers & Bjiirkerud (1973).

(f) Sucrose grarliertt

centrifuiryatior~

Analytical sucrose gradient centrifugatjion IIUS pcrformctl as dcscribctl prc\,iousl\ (Dudai et al., 1972a). Apparent sedimentation coefficients (.T~,,~) \vt’re dctr~rmillrd t)\. comparison with the standard markor enzyme fl-galactosidase (16 8) atld cat,alasc (I 1.4 S\.

Gel filtration was performed on a Sepharose CL-2B columl~ (1.8 cm k 90 cm) at 4°C: iI1 0.5 mm-azide, 1 M-N&~, 0.01 M-phosphate (pH 7.0). The column was pre-washed wittl 0.1 o/0 bovine serum albumin in the elution buffer before sample applicatiotl. The exclusion volume (v,) was taken as the clution volune of bacteriophage T4, and tllr total gel bed volume (q) as the elution volume of K,Fe(CN),. Tllr relative exclusion volume, h’,,. is equal to (v - Ue)/(wt ~ w,), whore ?I is t,hc clutjiotl ~oluruc~ for a particular molecular species. Stokes radii of molecular forms of AC&Ease \vere est,imated according to Siegel $ Mollt>(1966), using a linear calibration plot of R, versus l/--log K,, obtained using startdart protein markers which included rabbit skeletal muscle myosin (IZ, 2 215 A%), fibrhlogrrl (107 A), fi-galactosidase (82 A) and catalase (52 !I) (see Bon et al., 1973,197A). Molecular weights were estimated from t,llr lineal relationship between molecular weight and R,*s (Siegel & Monty. 1966), assuming similar partial specific volumes for the for different AcChEase species (Bon et al., 1976), and losing the same prot,ein markers calibration. (h) Acrylamide

gel electrophoresis

SDS/acrylamide gel electrophoresis on cylindrical gels was performed according t)o Shapiro et al. (1967), but gel concentrat’ions of 3.1?b acrylamide and O.O89o/o bisacrylamidr were employed in order to permit migration of high molecular weight component,s. Markers employed included /3-galactosidase and bovine serum albumin, as well as rabbit skeletal muscle myosin oligomers prepared by cross-linking of monomeric myosin wit Ir dimethyl suberimidate according to Reisler et al. (1973). SDS/acrylamide gradient slab gels were prepared according to O’Farrell (1975), 51’ to 15qb or 50/;, to 20% acrylamide gradients being routinely employed. Protein markers utilized included bovine serum albmnill. tltbulirr, ovalbumin. lact,atc dehgdrogenasr. trypsin and lysozyme. Both cylindrical and slab gels were stained with Coomassic Brilliarlt Bh~e K. (:(*I scanning was performed in a Gilford 2400-S spectrophotometer at 560 nm. Homogenates in wllich the catalytic sites of AcChEase had been labeled with L3H]diisopropylfluorophosphate were electrophoresed in 3.1?/, acrylamide gels, as described above. Gels were sliced and monitored for radioact,i\-ity as described previousl\(Dlldai & Silman. 1971). For determination of tile amino acid composition of tile different proteilr bands appoaring on the acrylamide gels after electrophoresis, the gels were sliced and slices containing the appropriate polypeptide components were taken for analysis. These slices were eittrer directly hydrolyzed in 6 N-HCl under the conditions normally used for amino acid analysis or, alternatively, the slices were homogenized manually in 0.01 M-phosphate buffer (pH 7.2), and the supernatants from low-speed centrifugation were taken for acid hydrolysis and amino acid analysis as described above.

3. Results (a) Elongated forms

of acetylcholinesterase

The experiments described below utilized purified preparations of elongated forms of AcChEase, extracted at high ionic strength from fresh electric organ tissue and

STRUCTURE

OF

“97

ACETYT~CHOLINESTERASE

purified by affinity chromatography (Dudai et al., 19726). These purified preparations contain the two principal elongated molecular forms. 18 S a,nd 14 S AcChEase. as shown in Figure l(a) (solid line), and maintain their known capacity to aggregate reversibly at low ionic strength (Fig. l(b), solid line) (Massoulie et al., 1970: Dudai et al., 19726). As will be apparent in the following sect’ions, the complex structure of the elongated form of AcChEase is partially maintained by interpolypeptide chain disulfide bonds. In order to eliminate the possibility that these structures are artefacts caused 1-q

0.4

0.2

E c N ? d 6 B. .‘-.?

0.4

1 0.2

:: % w” 6 8

(e) 20.6 S

0.4

0.2

18.6s

;I 1I I I , I I ! I i /

'

\

I Fraction

FIG. 1. Action of trypsin and collagenasc on purified clongat,od forma of eltMric eel acetylcholine&erase. Sucrose gradient centrifugation was performed on .50/b to 20% gradients, containing either 1.0 iv-NaC1, 0.01 M-phosphate (pH 7.0) ((a), ( c ) and (e)) or 0.1 x-NaCl, 0.01 &r-phosphate (pH 7.0) ((b), (d) and (f)), before and after (-~----) digestion with either trypsin or (---) collagenase. Digestions were performed in 0.2 M-NaCl, 0.05 M-Tris (pH 7.0), at 25°C for 1 h. (a) and (b) 14 S + 18 S AcChEase (1 mg/ml) with trypsin (50 pg/ml). ( c ) and (d) 14 S + 18 S AcChEase (400 pg/ml) with collagenase (20 &ml). ( e ) and (f) 1X S AcChEase (100 pg/ml) with collagenasc (10 x+$4.

“98

IA. ANC:I,LS’I’EH

.iNI)

I.

HI I,.u.lN

oxidative cross-linking of free sulfhydryl groups on different polypcptide chains exposed during the process of extraction and purification. as has been shown to ocou~‘. for example, in t’he CRW of the rnurinr (H-2) Iiistocompat,il)ilit~. antigen (Hrrlnin~ et aZ., 1976). the following control experiment was performed: extracts of fresh electric organ tissue were prepared by homogenization in a tnedium containing eithrl iodoacetic acid or iodoacetamide, each at three different concentrations, i.e. ~02 III. 0.1 M and 0.2 M, as described in Materials and Methods. Even at the highest concentration (0.2 M) of the alkylating agents, no decrease in t’he AcChEase activity of thest extracts was observed relative t,o the control. On sucrose gradient centrifugation. t,htb same pattern of molecular forms was detected in the samples treated with the alkylat,ing agents as in controls. i.e. the two major 18 S and 14 S species seen in Figure l(x) appeared in similar ratios, and still aggregated reversibly at low ionic strength. Since both reagents were present at concentrations capable of blocking all free sulfhydryls exposed during the extraction procedure (see, e.g. Gurd, 1972). it can be concluded that the 18 S and 14 S elongated molecular forms are not artefacts produced hv formation of inter-polypeptide chain disulfide bonds during this process. (b) Digestion

of elongated .forrns qf acetylcholinesterase

Dy collagenase

Figure 1 compares the effects of collagenase and trypsin on purified elongated forms of electric eel AcChEase, as analyzed by sucrose gradient centrifugation at low (O-1 M-NaCl) and high (1 M-NaCl) ionic strength. Collagenase? like trypsin, abolishes the capacity of both 14 S and 18 S AcChE ase to aggregate at low ionic strength (compare Fig. l(d) and (f) with Fig. l(b)). H owever. under the conditions employed the two enzymes yield very different products. Both 18 S and 14 S AcChEase are converted by trypsin almost ent’irely to the 1 I S species (Fig. I (a’) and (b)), whereas on collagenase treatment ea,ch one yields a single non-aggregating molecular form of sedimentation coefficient significantly higher than that of the intact’ non-aggregating form (Fig. l(c) to (f)), and little or no 11 S enzyme. Thus 18 S “aggregating” AcChEase yields 20 S “non-aggregating” AcChEase (Fig. l(e) and (f)) and 14 S aggregating

FIG. 2. Determination of the Stokes radius (22,) of 20 S AcChEase, produced by collagenase digestion of 18 S AcChEase, by gel filtration on a Sepharose CL-ZB column. The linear calibration plot of R, IJWSUB d-log K,,, where K,, is the relative exclusion parameter, was obtained by gel filtration of standard protein markers as described in Materials and Methods. The ranges spanned by the double arrows represent the scatter of results of 3 to 5 experiments.

STRUCTURE

OF

“99

ACETYLCHOLINESTERASE

0

ff..s (lo-’ 8c3 FIG. 3. Molecular weight estimation of 18 S AcChEase and of the 20 8 AcChEase produced from it by collagenase digestion. Molecular weight values were obt*ained using the linear relationship between the molecu1a.r weight and the product of the St,okes radius and sedimentation coefficient, (R;s), as determined from gel filtration (Fig. 2) and sucrose gradient aentrifugation (Fq. 1). respectively. The standard calibration curve was generated using the same protrins rmployod fol calibration of the gel filtration column (see Materials and Methods). TABLE

Molecuhr Molecular form of AcChEasr

parameters

Apparent sndiment,ation coefficient,

1

of different ,forms qf acetylcholinederase St>okcw radius,

Molecular weight

R,

%PP (S)

(a

18 8

18.4&0.3

14243

2l370*90

1,040,000

+ 80,000

“0 s

-“0.3*0.5

11513

2350 + 60

940,000

+ 50,000

11 s

11.3*09

8613

960 + 30

360,000

c “0,000

AcChEase analogously yields 16 S non-aggregating AcChEase. Collagenase action can he efficiently inhibited by l,lO-phenanthroline. a known inhibitor of t.his enz,yme (Reifter & Harper, 1971). Gel filtration shows that collagenase conversion of 18 S AcChEase to 20 S AcChEasc is accompanied by a reduction in Stokes radius (R,) from 145 A to 115 8, as shown in Figure 2. From the Stokes radius (R,) and the sedimentation coefficient (s). it was possible to estimate the molecular weight of 20 S AcChEase, a,s well as 18 S AcChEase: from a plot of molecular weight versus R;s (Siegel & Monty, 1966; Bon et nl., 1973). nsing a calibration curve generated with suitable markers (Fig. 3). Table 1 shows that conversion of 18 S to 20 S AcChEase involves only a small decrease in molecular weight, which, taken in conjunction with the large decrease in Stokes radius, indicates that colla.ganase markedly reduces the asymmetry of the AcChEase molecule. (c) Amino

acid composition

Table 2 shows that preparations of 14 S + 18 S AcChEase have a similar overall amino acid composition to the 11 S AcChEase obtained subsequent to tryptic dipes-

300

Lys

4-4+0.2

4.4 j: 0.4

His

2.350.1

2.5-1:0.2

Arg

5.3f0.5

Asp Thr

12.6hO.7

11.4 to.9

4-l&0.2

4.3+0.1

S0r

7.910.9

7~9&0.3

Glu

10~8~OG3

10.9 LO.6

PlW G1.v

7-l kO.9 8.450.8

7.1$_0.7 11.3 10.6

Ala

6.450.5

6.110.4

Val

6.510.6

ti,BmiI0.7

6.4 + 0.2

Met

3.2&

1.6 to.9

2.8 + 1.0

Ile

3-7&0.3

3.3 to.2

3.4 to.4

L0U

9.1 +O.S

tK,t0.5

9.6 ‘r0.5

TY~ Phe

3.2+04

3.0 tro.:3

5.5&0.7

4.8+0.5

2.7 to.6 5.:j l-O.-”

JW


HYP

5.1 I 0.3

1.0

0.10+0.06

6.3+0.2

O-60 iO.15

0.x1 +0.02

O+i3fO.13

w:<4 iI 0.05

Values are expressed as moles per 100 moles of t,otal amino acids recovered 1~ s.c.m. ; averaged for 9 analyses of 11 S AcChEase, 7 analyses of 14 S 4~ 18 8 AcChEase and 3 analysts of 16 R 20 S AcChEase. Hydroxylysine was analyzed in 5 samples of both 14 S + 18 S .-\cChEase and 11 H AcChEase and in 3 samples of 16 S + 20 S AcChEaxe. Hydroxyprolino was analyzed colorimetritally in 8 samples of 14 S + 18 S AcChEase, 6 samples of 11 R enzyme and 3 samples of 16 S {- PO S AcChEase.

tion. However, 14 S -+ 18 S AcChEase contains significant amounts of both hydroxyproline and hydroxylysine, which are almost completely absent in 11 S AcChEase. and also has a significantly higher glycine content. In order to see how collagenase treatment affected the amino acid composition of the elongated forms of AcChEase, samples of 14 S + 18 S AcChEase were treatfed with collagenase; after complete conversion to 16 S + 20 S AcChEase had been demonstrated by sucrose gradient centrifugation, the product was adsorbed on a phenyltrimethylammonium-Sepharose resin of the type routinely used for purification of 11 S AcChEase (Dudai et al., 1972a). After extensive washing of the column, the collagenase-modified enzyme was eluted with 10 mM-decamethonium. The righthand column in Table 2 shows that collagenase treatment led to a reduction of approximately 50% in the hydroxyproline and hydroxylysine contents of the enzyme, and to a glycine content intermediate between that of 11 S AcChEase and 14 S -+ 18 S AcChEase. (d) Polypeptide

components of elongated forms qf acetykholinesterase and after collagenuse digestion

On SDS/polyacrylamide

gel electrophoresis

in the absence of reducing

before agent 11 S

STRUCTURE

OF

301

ACETYLCHOLINESTERASE

(b)

(d)

460 360 290

165 160 I30

60

E’Ic:. 4. SlX3/polyscrylamide gel electrophvrwis of 14 S $ 18 S AcChEase, m the absence of reducing agent, before and after collagenase digestion. Electrophowxis was performed on 3.19; gels as described in Materials and Methods, and staining was with Coomassie Brilliant, Blue R. (a) 14 8 + 18 S AcChEase, control. (b) 14 S + 18 S .-\cChEaw after collagenave treatment, tligest,ion performed as in Fig. l(e). (c) Purified collagenanr utilized in the digestion experiment. (d) 11 S AnChEasr. Molecular weights are shown Y IO- 3.

ACChEM! reveals, as its two principal components, a dimeric species (approx. 160,000) and the approximately 80,000 monomer (Fig. 4(d), Fig. 5(d)). In 14 S + 18 S AcChEase little or no monomer is observed, the dimer accounts for only 50 to 609, of the polypeptide chains, and the remaiuder move as much heavier species, which consist primarily of two discrete bands (Fig. 4(a): Fig. 5(a)). Their apparent molecular weights can be estimated as approxima.tely 360,000 and 460,000 bg calibration with mgosin oligomers prepared according to Reisler et al. (1973). If 18 S and 14 S AcChEase are electrophoresed separately, it can be seen that the 460ZOO0 component is derived exclusively from 18 S AcChEa.se and the 366,000 component from 14 S AcChEase. In both cases the dimer is also present,. accounting for 50 to 600/;, of the total protein (Fig. 5(b) and (c)). Similar results have recently been reported by McCann & Rosenberry (1977). Tf [ 3H]diisopropylfluorophosphate-labeling experiments were performed on the electric organ extracts obtained by homogenization in the presence of alkylat.ing agents (see above), electrophoresis revealed the same radioactive peaks seen previously (Silman & .Dudai, 1975), corresponding in mobility and relative ratios to the peaks obtained by cblectrophoresis of purified 14 S + 18 S AcChEase stained for protein. Thus blocking of free sulfhydrvl groups by iodoalkylation not only causes no change in the sedimentation properties of 18 S and 14 S AcChEase, but also causes no appreciable change in the polppeptide profile on SDS/polyacrplamide gels run in the absence of reducing agent. The presence of collagen-like polypeptides in the 360,000 and 460,000 molecular weight components was directly examined by extracting them from the acrylarnide gels and determining their hydroxylysine content. Although this procedure permitted

L-

360 165

;

0.0

A

r L 1

,

460

rn

0.4

0.0

1 J

,“I 160

0.8 165 n

t

-

PIG. 5. IIensitometer tracings of SDS/polyacrylamide gel electrophoretograms samples of AcChEase elsctrophoresed under the conditions described in Fig. 4. (a) AcChEase. (b) 14 S AcChEase. (c) 18 S AcChEaso. (d) II 8 AcChEaso. (e) 14 S + 18 (400 pg/ml) incubated with collagenase (20 pg/ml) for 1 h at 26°C in 0.2 nT.NaCl, 0.01 (pH 7.0). (f) 14 8 + 18 8 AcChEase incubat,ed with collagenase as in (R) but in the 5 mxr-l,lO-phenanthroline. Molecular weighk indicakl x lo-“.

of purified 14 S + 18 S 8 AcChEaae wphosphattb presence of

only yualitative analysis, it clearly demonstrated that the heavy components contain significant amounts of hydroxylysine. Thus a combined extract of the 360,000 and 460,000 bands had a hydroxylysine content of 0.6 to 0.7:/,, while the 160,000 component contained no detectable hydroxylysine. When 14 S + 18 S AcChEase is treated with collagenase, the two heavy polypeptide components originally present vanish, and are replaced by two new species with apparent molecular weights of approximately 290,000 and 185,000 (Pig. 4(b), Fig. 5(e)). However, electrophoresis of 14 S and 18 S AcChEase separately, after collagenase treatment, reveals that they both contain these two new polypeptides, neither of them

S’t’RlJCTURE

OF

:<(I:?

i~CE’I’Tfl,CHOLINES’1’ER;\SE

bGng derived exclusively from the products of collagenase digestion of either 18 S 01 I4 S AcChEase. Incubation of 14 S + 18 S AcChEase with collagenase in the presencr of l,lO-phenanthroline (Fig. 6(f)) reveals that it retards digestion of the heavy polypeptide components of 14 S + 18 S AcChEase, as might be expected from its in hi bitory ~+Fect on collagenase digestion as monitored on sucrose gradients. Rosenberry & Richardson (1977) reported that two polypeptidea of apparent molecular weight 41 .OOOand 44,000 are present in 14 S 1 18 S ScChEase and absent it1 11 S AcClhEase. They suggested that these polgpeptides might be the putative collagen pol,vpept!ides of the tail, on the basis of the difference in amino acid composition bet~wecw the catalytic subunit dimers and oligomers. In agreement with t’hcw authors. w observe two such components (of apparent molecular weight 45.000 and (b)

Cc)

Cd)

FIG. 0. Sl)S/polyac~~ylttmid~ gel electrophort:sis of Ii S f IX S .k(‘hEase, in thn preemce of reducing agent. before and after cvllagenase digestion. E:lrctrophowks was pwfortned on 5”Yo t,o 200/ acrylamide gradient slab gels, in the presence of 8-m(:~captl)et,harlol, a~ desribed in Material* and Methods. Staining was with Coomassie Brilliant Blue H. (a) 14 S +- 18 S AcChEa;r. (b) and (c) Samples of 14 S + 18 A AcChEase incubated with collagenitw :ti in Fig. l(e) for 30 min and 2 h, rt*spectively. ((1) 11 R AcChEasr. Molecular weights s’ IO- ‘j.

47,000) in 14 S + 18 S AcChEase on electrophoresis in the presence of j3mercaptoethanol (Fig. 6(a)), b u t not in its absence, and do not detect them in 11 S AcChEase (Fig. 6(d)). These bands are greatly diminished by collagenase treatment, leading to conversion to 16 S + 20 S AcChEase (Fig. 6(b) and (c)), and on electrophoresis in the presence of /3-mercaptoethanol their disappearance is accompanied

304

I,.

As(:1~l8’1’lcli

.iSI)

I.

SII,~i.\S

by the appearance of a WL\V approximat,ely 30.000 spcGeh (Fig. pi and ((.)), a11c1~ILs(, of smaller polypeptides of appawrlt ~trolt~t~r~lat~\\ t,ight ~rpproxiiklatt~l~~ IO.000 to 13.000. (e) Ejjfect

qf trypitt ott the

yolype~~fitlr

prqjil~s

c!/ dotqdcrl

jiwttts

O/

rtcefylcholitrueferasr

The data presented above show that both in 14 8 $- 18 S XcChEase autl in thtb products of its collagenase dig&ion all polypeptide componenls of the enzymc~ migrate on SDS/polyacrylamide gels as dimtbrs or heavier species in the abst>nce of reducing agent. However, samples of I1 S .\cChEasr~. purified subsequently to tryptic digestion, often conta,ined quite a large proportion of the 80.000 monomeric species even in the absence of reducing agrnt (set,. e.g. Fig. l(d)). Since thtl prrrist site of action of trypsin is of import’ancc for undersbanding the mode of att~achmrnt of the collagen tail to the catalytic subunits. \ve havcl investigat,cd the effect of tjrypsiu on 14 S + 18 S AcChEase in greater detail. Figure 7 shows t’hcx effects of’ varying amounts of trypsin on 14 S + 18 S AcChEase as studied by c+ctrophorrsis wit’h and without p-mercaptoethanol. Under the conditions employed, trypsin. as is alread). known, converts the 460.000 and 360,000 species to the 160.000 dimc~r. wit’h transient appearance of an approximately 170.000 species. However. substqutbnt to thtx disappearance of the 360,000 and 460,000 forms. the 160.000 specic>s begins to diminish and is replaced by the 80.000 component. even though no reducing agent is prcsrnt, (Fig. 7(b) to (d)). Thus at, the highest t)rypsin concent’rat8ion tmployed. the XO,C)OO species comprised over 6Oqb of the total material. and t,he 160,WU dimer became the lesser component (Fig. 7(d)). The gels run in the presence of P-nlercaptoethailol (Fig. 7(e) and (f)) show that under these conditions no significant conversion O~WWS of the 80,000 polypeptide to the 60,OW and 20,000 species which are produced I)) t’rypsin or by autolysis (Dudai & Silman, 19746 : Rosenberry it al.. 1974: Morrod et ~1.. 1975). Thus the tryptic conversion of the 160,000 dimer to tllc 80,000 monomer. in the absence of reducing agent. is an tavcntj which precedes this latter cl~vapc~ of the 80,000 polypeptide. forms of’ AcCliEase. The polypeptide composit’ions of the various molecular obtained t)y SDS/polyacrylamide gel elect’rophoresis under reduc*ing and noll-reducing conditions, as described above. are summarized in Table 3.

Principal

of d(ffewnt molecular jmtls of polypeptide compotte~~ as visualized b?y SIW/acrylarnide gel electrophoresis

acetylcholinesterase

Mdecular form

Hoforr

I’olypeptido species (M, X 1W3) After reduction PRdUCtioII

18 s

460,

lti0

80, 47, 45

14 s

360,

160

x0, 47, 45

STRUCTURE 0.6

OF

;ZCETYLCIHOLINESTERhRE

’ (a)

i

162

(b)

160

’

52

32 I

I

0.8

o-4

0.0

I’IG. 7. Effect of trypsin on the polypeptide pattern of purified 14 S I- 1X k-5AcChEase. SDS gel ~:lrc:t~ophor~?sis was performed on cylindrical 3’ 1 Td, acrylamide gels, in the absence of sulfhydryl slab gels, in thu presence of rctlucing agent) ((a) to (d)), and on 5% to 16% acrylamide gradient ,%mercaptorthanol ((e) and (f)). Staining and densit~ometer treeing were performed as described in Materials and Methods. 14 S + 18 S AcChEaae (1 mg/ml) was incubated for 1 h at, 25°C with various trypsin concentrations. (a) and (e) 14 S + 1X S AcChEaw control. without trypsin; (b) with 1 pg t,rypsin/ml; (c) wit,h 6 pg t,rypsin/ml: (d) and (f) with 32 pg t,rypsin/ml. Molecular weights x 10. 3.

4. Discussion The physicochemical data and electron microscopic observations on t*he elongated forms of EEPctrophorus AcChEase suggested the presence of one, two and three tetramers in 9 S, 14 S and 18 S AcChEasr. respectively. with a tail of similar dimensions in all three species (Dudai et d., 1973: Rieger et nl., 1973; Bon at al., 1973). These data are consistent with a model in which each tetremcr is attached bo one stra,nd of

FIa. sulfate,

8. Model of the 18 S AcChEase trypsin and collagenme.

molecule

showing

points

of cleavage

by sodium

dotloc,yl

a collagen triple helix, as shown in Figure 8, and electron micrographs by Cartaud et al. (1975) suggest a physical basis for this model. The amino acid composition data and the specific effects of collagenase on the elongated forms of ilcChEase strongly support such a structure, as discussed below. as does immunochemical evidence demonstrating cross-reactivity between collagen and elongated forms of AcChEnse (L. Anglister, I. Silman, S. Fuchs & R. Tarrab-Hazdai. manuscript in preparation). Recently, Johnson et al. (1977) demonstrated that puritied collagcnase could attack the elongated forms of AcChEase in extract,s of electric organ tissue, converting each one to a new form of significantly higher sedimentation coefficient. Our sedimentat,ion coefficient values for the products of digestion of purified 14 S and 18 S AcChEase by collagenase (Fig. I(c) to (f)) are similar t,o those reported by Johnson et al. (1977) for the salt extracts, and we also show that this conversion occurs without significant concomitant formation of 11 S AcChEase. However, while they reported that modification by collagenase did not affect the characteristic tendency of t,he elongated forms of AcChEase to aggregate reversibly at low ionic strength. we clearly demonstrate that collagenase digestion does destroy this property (compare Fig. 1 (d) with (1))). The discrepancy between our results and those of ,Johnson rf al. (1977) may be due t#o the fact that they were working with salt extracts, while we were studying thcx purified enzyme. Our centrifugation and gel filtration measurements (Figs I to 3, Table 1). takcw together with the amino acid analyses (Table 2). strongly suggest that collagenase cleaves the tail at a specific point about half way down the collagen sequence, producing a more symmetrical species in which the residual tail still holds together all three tetramers (in the 20 S form) or two t’etramers (in the 16 8 form) by covalent OI non-covalent bonds (see below). although it’ lacks the segment, responsible for aggregation at low ionic strength. Electron microscope observations (Dudai et al.. 1973; Rieger rt al., 1973; Cartaud et al., 1975) indicate that the tail in the elongated forms of AcChEase is about 500 a long. Since there a,re about 100 amino acid residues in a 100 A segment of a, collagen triple helix (Traub & Piez, 1971), if the tail were indeed a single triplo-helix, it, would thus contain three strands, each of molecular weight approximately 17,000, giving a

STRUCTURE

OF ACETYLCHOLINESTERASE

307

total molecular weight of approximately 50,000, which is in the range to be expected from physicochemical measurements (Dudai el aZ., 1973 ; Bon et al., 1973,1976 : Silman & Dudai, 1975). The hydroxyproline content of 14 S -+ 18 S AcChEase would be in agreement with such an entity if the hydroxyproline content of the tail was similar to the high values observed in mammalian basement membranes (Kefalides, 1973a; Gelman et al., 1976). It is of interest that in 14 S + 18 S AcChEase the ratio of hydroxylysine to hydroxyproline is unusually high, and that such a high ratio is also characteristic of basement membrane collagen (see, e.g. Kefalides, 19736). The results discussed so far support both the general model shown in Figure 8 and the putative role of the tail in association of AcChEase with the basement membrane. The results of SDS/polyacrylamide gel electrophoresis experiments, in the presence and absence of reducing agent, provide clear evidence that disulfide bonds are involved in subunit-subunit and subunit-tail interactions, and probably also in interactions between the strands of the tail. The experiments in which electric organ tissue was homogenized in the presence of alkylating agents seem to rule out the possibility that these disulfide bonds are artefacts of the extraction process, a possibility raised t)J Bon & Massouli& (1976b). The elongated forms of AcChEase. as well as the subunit dimers and putative subunit-tail complexes observed on t#he gels. thus represent, intrinsic structures which are present in the intact t’issue. The presence of the heavy 460,000 and 360,000 components observed on acrylamidtx gels in 18 S and 14 S AcChEase, respect,ively. but not in 11 S AcChEase, which is devoid of the tail, suggested that these heavy species represent tail-head complexes linked by disulfide bonds. The results of t’wo Oypes of experiment provided strong evidence that this is indeed the case: the detection of hydroxvlysine in these t)wo components but not in the 160,000 dimer (see also Rosenberry $ Richardson, 1977). and the specific modification of both the 460,000 and 360.000 species by collagenase. The SDSjacrplamide gel electrophoresis patterns in the absence of reducing agent are thus best, accounted for by a model in which half the cat~alytic subunits in t,hrx elongated forms of AcChEase are present as dimers. not covalently attached to t,hc collagen tail. while the other half are present as dimers. which are covalently attached to the tail. again presumably by disulfide bonds. Unless the collagen strands do not dissociate on denaturation with SDS, which is unlikely (Furthmayer $ Timpl. 1971). it must also be assumed that these strands are linked by disulfide bonds in order to explain the dimensions of the entities observed on the SDS/polyacrylamide gels prior to reduction. The apparent molecular weights of the 460.000 and 360.000 components. derived from 18 S and 14 S AcChEase, respectively. must be considered approximations. since such complex structures. involving several interchain disulfide bonds. should not be expected to migrate exactly like single polypeptides of similar molecular weight (see. e.g. Griffith, 1972). Nevertheless, it is plausible that t’he 460,000 component corresponds to six catalytic subunits linked to the collagen tail. and t,he 36O.OO(b component t)o four subunits linked to the tail. The appearance of the two species produced by collagenase digestion of both 18 S and 14 S AcChEase. i.e. species of apparent’ molecular weight 290,000 and 185.000, i s most easilg rationalized I)> assuming that certain interchain disulfide linkages in the tail are present in t’he segment removed or digested by collagenase. They may t,hus represent. a subunit dimer bound to one strand of the residual tail (185,000)~ along with a species in whirh two subunit dimers are attached to two rollagen strands linked by disulfide bonds st,ill present in the residual tail (290,000).

30x

I,.

;\N(:I,IS’I’ER

ASI)

I.

SlI,ST‘-\S

In agrccrment with the observations of Rosenberry 8r. Richardson ( IHii). \\I’ ww sistent1.v detect. in 14 S .*- 18 S AcChEase. two polvpeptides. of apparent molecula.t weights 45.000 and 47,000, on SDS/polyacrylamide gels in t,hrh presence of reducing agent. but not in its absence. The fact, that collagenase preferentially digests thcstl components. under the conditions in I\-hich it produces 16 S * 20 S ArChEasca. provides t*he first direct’ evidence that these polypeptides contain collagen-likes sequences. The new major 30.000 pol.ypept’ide which appears after collagrnasc digestion is presumably derived from the residual tail since it. t)oo. is visualized only in thr presence of reducing agent. It’ should be noted that the true molecular weights of collagen polypeptides appear t,o be considerably lower than t,he apparent’ values estimated from calibration curves for SDS/polyacrylamidr gel electrophoresis genrrated with globular marker proteins. this discrepancy being particularly marked for small polypeptides (Purt’hmayer & Timpl. 1971). Thus. components of apparent’ molecular weight approximately 45,000 might represent collagen-like polypeptides of approximatel,v 30.000. giving an overall value of 60.000 for A triple helix. which is in the range to be expected for t,he total mass of the t’ail. The approximately 30.000 component appearing after collagenase digestion might have a t,rur molecular weight as low as 12.000. as might be expected from the phgsicochemical and amino acid a,nalysis data suggesting that about half the tail is removed by collagenasc digest,ion. The final point to be discussed is the mode of attachment of th(h catalytic subunit t)etramers to the tail. The data from SDS/polyacrylamide gel electrophoresis clearly indicate that, while half the dimers are not covalent.l,y attached to thr tail, the remaining subunits are linked to it by disulfide bonds. In order to furt,her investigate this point we examined the 11 S species obta,ined by trypt’ic digestion of purified 14 S 4 18 S AcChEase. The experiment shown in Figure 7 demonstrates that tryptic digestion of 14 S + 18 S AcChEase yields a product, in which somewhat over .5Oq,, of t,hr catalytic subunits migrate as monomers on SDS/acrylamide gels even in the absenct of reducing agent. while the 80,000 polypeptide i&elf is not significantly cleaved t’o the 60.000 and 20,000 fragments which appear in large amounts in 11 8 AcChEast after autolysis (see Fig. 6(d)). Th ese results suggest that the disulfide bonds involved in intersubunit linkage are close to the end of the catalytic subunit polypept’ide, a possibility which is supported by the report of Ron & MassouliP (1976a). who isolated an enzymically active 80,000 monomeric form of AcChEsse from autolysed electric organ tissue. It is thus possible to obtain, by tryptic digestion, 11 S tetramers differing somewhat from those purified from autolyzed tissue, which contain primarily subunit dimers (Rosenberry, 1975: Rosenberry & Richardson. 1977). Rosenberrp and his co-workers (Rosenberry, 1975) have provided evidence that in 11 S AcChEase purified from autolyzed tissue only one sulfhydryl group per catalytic subunit is involved in interchain disulfide bond formation, and have suggested (Rosenberry & Richardson. 1977) tha,t in those subunits which are covalently linked to the tail. this sulfhydryl is used not for subunit-subunit disulfide bond formation, but for linkage to a sulfhydryl group on the tail polypeptide. This would mean that in 11 S AcChEase two types of dimer exist. in one of which the subunits are linked via a fragment of thtt tail. Our data are not inconsistent with such a model. which might’ also help to explain wh,v not all the catalytic subunits in 18 S AcChEase are readily converted to monomers by tryptic digestion. Moreover, we now have evidence. based on SDSlacrylamidc gel electrophoresis of mixtures of 14 S t- 18 9 AcChEase and 1I S AcChEase la beled \vit,h respect)ively. showing that, there art[“HI- and 114Cldiisopropylfluorophosphate,

STRUCTURE

OF ACETYLCHOLINESTERASE

309

indeed two distinct dimers (Anglister & Silman, manuscript in preparation). However, the clear differences observed between the subunit I&terns of 11 S AcChEase preparations obtained by autolysis and by tryptic digestion suggest that it will be necessary to carry out direct reduction and alkylation st,udies on 18 S AcChEase in order to clarify the number and location of the sulfhydryl groups involved in intersubunit and tail-subunit disulfide bond formation in the intact molecule. In certain respects the structure of the elongated forms of AcChEase resembles that of factor Clq from complement. However. there is one major difference: whereas Clq contains globular sequences and collagen-like sequences which are part of the samt’ polypeptide chain (Reid $ Porter, 1975), our data show that, in AcChEase t’his is not the case. It) is thus possible that the two polypeptide components are biosynthesized separately and either assembled intracellularly and then secreted. or seer&d separateI> and assembled extracellularly. A second possibility is that the two components arc synthesized by different cells, e.g. BcChEase by the presynapt,ic n(‘rve cell and collagen b), the rlectroplax. and that assembly occurs extraccllularly. X third possibilit,y is bhat the AcChEase-tail complex is produced by protrolytic cleavage of a sin& pre-existing polypept,ide chain analogous to pro-collagen. thta product, of this cleavagc~ being held together by disulfidc bridges. In this corm&ion. it should be noted t,hat disulfide bridges are characteristic of the COOH-terminal region of procollagen (Byers et al., 1975) and have been shown to link globular and filamentous components of basement membrane collagen (Olsen et nZ., 1973) : moreover. basement nlrmbran(~ collagen seems to resemble procollagen in certain respects (Minor rt al.. 1976). The data cited in the Introduction on the effect of collagenaxe on neuromuscutaI preparations and on the association of specific elongated forms of AcChEase with thtb muscle endpl&e. suggest that the functional AcChEasch in skcl&ll musctt~ synapses resembles electric organ AcChEase in its molecular structure. and is anchored in the synaptic cleft’ in a similar fashion. The levels and distribution of AcChEase in tIlta synapse u-ill obviously control the level of acetylcholinr in the synaptic cleft. Furtht~r studies on the sbructure and mode of assembly of t)he txtongatcd forms of t~lcctric organ AcCh Ease and similar studies on skeletal muscle AcChEase are thus of irnportance for understanding the role of the t~nzyme during synaptoyencsis and in t tw functioning synapse.

This research ~vas supported by grants from t,he Muscular Dystroplly Associatiolls of America, t,hr United States Israel Bina.tional Science Follndatiorr and the Israel Comlnittw for Basic Research. We thank Mrs Esther Roth for expert technical assistaricr. Dr Yaditi Dudai for his participation in some of the early oxprriments, and Dr Sara Fuclls, Dr Shmaryahu Blumberp. Dr Yadin Dudai and Dr Vivian ‘l’eichherq for ttlcsil critical readirlg of the manuscript. REFERENCES Anglister, L., Rogozinski, S. & Silman, I. (1976). FEBS Letters, 69. 12%132. Betz. W. J. h Sakmann, B. (1971). Nature New Riol. 232, 94 95. Betz, W. J. &. Sakmann, B. (1973). J. Physiol. 230, 673-688. BOII, S. & MassouliB, J. (1976a). FEBS Letters, 67, 99-103. Bon, S. & MassouliB, J. (1976b). FEBS Letters, 71, 273-278. Bon, S., Rieger. F. & MassouliB, J. (1973). Eur. J. Biochem. 35, 372-379. Ron, S., Huet, M., Lemonnier, M., R,ieger, F. & Massonlih, .I. (1976). Eur. .i. Riochem.

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I. HI I,ivl.AS

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RTRUCTURE Silman, I., Anglister, L. Muscular dystrophy Press, New York. Spackman, D. H., Stein, Traub, W. 8z Pica, K. A.

OF ACETYLCHOLINESTERASE

31 1

& Gazit, H. (1978). In Biochen&ry of Myasthenia gravis and (Lunt, G. G. & Merchbanks, R. M., eds), pp. 119--l 29. Scademic W:. H. & Moore, 8. (1958). dna.l. Ch,em. 30, 1190- 1206. (1971). Advan. Protein Chem. 25. 24% 352.