8
Eur. J. Biochem. 68, 523-530 (1976) Molecular Forms of Electrophorus Acetylcholinesterase Molecular Weight and Composition Suzanne BON, Marianne HUET, Marguerite LEMONNIER, Franqois RIEGER, and Jean MASSOULIE Laboratoire de Neurobiologie, Ecole Normale Suptrieure, Paris, Laboratoire de Physique Biologique, Institut de Biologie Molkulaire, Universite Paris VII, and Centre de Recherches sur les Proteines, Facultt de Medecine Lariboisikre-St Louis, Paris (Received March 20/July 22, 1976) Molecular weights for the series of six Electrophorus acetylcholinesterase forms have been determined either by the sedimentation-diffusion equilibrium method or, particularly in the case of the very scarce G’ and G” forms, from their Stokes radius and sedimentation coefficient values. Both methods are in excellent agreement. The results provide good evidence for the model previously proposed, G”, G’ and G containing one, two and four subunits, whereas A, C and D possess, in addition to respectively one, two and three tetrameric sets of such subunits, a structural element, the tail. Although the amino acid composition of ‘tailed’ and globular forms did not reveal any significant feature of this element, its mass, about 100000 daltons, could be deduced from a com- parison of molecular weights for the two classes of acetylcholinesterase forms. This value is in close agreement with electron microscopic data. The tail is thought to consist of three 30000-dalton strands. In our previous studies we have shown that acetyl- cholinesterase from Electrophorus electricus electric organ exists in a number of distinct molecular forms [ 1,2], which differ markedly in their physical chemical characteristics. Three forms, D (18.4 S), C (14.2 S) and A (9 S), behave hydrodynamically as asymmetric particles [3,4] and are seen in the electron microscope to possess an elongated ‘tail’ [5,6], which the three other forms G (11.8 S), G‘ (7.7 S) and G” (5.3 S), are lacking. An analysis of their subunits by electro- phoresis in sodium-dodecylsulfate polyacrylamide gel led us to the conclusion that all six forms contain iden- tical catalytic subunits (Bon and Massoulit, unpub- lished results). The catalytic properties of the small- est, which will be shown to be a monomer of the cata- lytic subunit, have been reported elsewhere [7] ; except for minor quantitative differences, they were found similar to the common enzymic properties of the poly- meric molecules. We have already proposed a model for the struc- ture of the acetylcholinesterase multiple forms [5], which is consistent with the hydrodynamic data [4], the electron microscopic appearance of A, C, D, G and G’ [5,6] and the conversion sequence that we ob- tained by proteolytic degradation or sonication of these enzymes [1,2,3,8]. The D molecule is the most complex and all others can be derived from it; it is made up of a compact assembly of three tetramers, attached to the tail. The C and A forms are obtained by splitting off one and two tetramers. The globular forms G and G’ are respectively a tetramer and a dimer. This model has been substantiated by micro- graphs of D molecules in which three tetramers can be identified; in some cases the proximal part of the tail was seen to split up into three filaments, each of them apparently attached to one of the tetramers [6]. Comprehensive data on all forms of Electrophorus acetylcholinesterase were, however, lacking, so that the model was still partly conjectural. In this paper we report a comparative study of their amino acid and carbohydrate composition and of their molecular weights, determined by sedimentation-diffusion equi- librium centrifugation and, for the least abundant forms, by an indirect method, based on sedimentation coefficients and Stokes radius data. MATERIALS AND METHODS Acetylcholinesterase Preparations Acetylcholinesterase was obtained from Electro- phorus electric organ homogenates in 1 M saline buffer (1 M NaCI, 0.05 M MgCI2, 0.01 M Tris-HC1 buffer pH 7) as described elsewhere [9]. All buffers contained

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Page 1: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

Eur. J . Biochem. 68, 523-530 (1976)

Molecular Forms of Electrophorus Acetylcholinesterase Molecular Weight and Composition

Suzanne BON, Marianne HUET, Marguerite LEMONNIER, Franqois RIEGER, and Jean MASSOULIE

Laboratoire de Neurobiologie, Ecole Normale Suptrieure, Paris, Laboratoire de Physique Biologique, Institut de Biologie Molkulaire, Universite Paris VII, and Centre de Recherches sur les Proteines, Facultt de Medecine Lariboisikre-St Louis, Paris

(Received March 20/July 22, 1976)

Molecular weights for the series of six Electrophorus acetylcholinesterase forms have been determined either by the sedimentation-diffusion equilibrium method or, particularly in the case of the very scarce G’ and G” forms, from their Stokes radius and sedimentation coefficient values.

Both methods are in excellent agreement. The results provide good evidence for the model previously proposed, G”, G’ and G containing one, two and four subunits, whereas A, C and D possess, in addition to respectively one, two and three tetrameric sets of such subunits, a structural element, the tail. Although the amino acid composition of ‘tailed’ and globular forms did not reveal any significant feature of this element, its mass, about 100000 daltons, could be deduced from a com- parison of molecular weights for the two classes of acetylcholinesterase forms. This value is in close agreement with electron microscopic data. The tail is thought to consist of three 30000-dalton strands.

In our previous studies we have shown that acetyl- cholinesterase from Electrophorus electricus electric organ exists in a number of distinct molecular forms [ 1,2], which differ markedly in their physical chemical characteristics. Three forms, D (18.4 S), C (14.2 S) and A (9 S), behave hydrodynamically as asymmetric particles [3,4] and are seen in the electron microscope to possess an elongated ‘tail’ [5,6], which the three other forms G (11.8 S), G‘ (7.7 S) and G” (5.3 S), are lacking. An analysis of their subunits by electro- phoresis in sodium-dodecylsulfate polyacrylamide gel led us to the conclusion that all six forms contain iden- tical catalytic subunits (Bon and Massoulit, unpub- lished results). The catalytic properties of the small- est, which will be shown to be a monomer of the cata- lytic subunit, have been reported elsewhere [7] ; except for minor quantitative differences, they were found similar to the common enzymic properties of the poly- meric molecules.

We have already proposed a model for the struc- ture of the acetylcholinesterase multiple forms [5], which is consistent with the hydrodynamic data [4], the electron microscopic appearance of A, C, D, G and G’ [5,6] and the conversion sequence that we ob- tained by proteolytic degradation or sonication of these enzymes [1,2,3,8]. The D molecule is the most complex and all others can be derived from it; it is

made up of a compact assembly of three tetramers, attached to the tail. The C and A forms are obtained by splitting off one and two tetramers. The globular forms G and G’ are respectively a tetramer and a dimer. This model has been substantiated by micro- graphs of D molecules in which three tetramers can be identified; in some cases the proximal part of the tail was seen to split up into three filaments, each of them apparently attached to one of the tetramers [6].

Comprehensive data on all forms of Electrophorus acetylcholinesterase were, however, lacking, so that the model was still partly conjectural. In this paper we report a comparative study of their amino acid and carbohydrate composition and of their molecular weights, determined by sedimentation-diffusion equi- librium centrifugation and, for the least abundant forms, by an indirect method, based on sedimentation coefficients and Stokes radius data.

MATERIALS AND METHODS

Acetylcholinesterase Preparations

Acetylcholinesterase was obtained from Electro- phorus electric organ homogenates in 1 M saline buffer (1 M NaCI, 0.05 M MgCI2, 0.01 M Tris-HC1 buffer pH 7) as described elsewhere [9]. All buffers contained

Page 2: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

524 Molecular Weight and Composition of Electrophorus Acetylcholinesterase

0.05 M MgCl, and 0.01 M Tris-HC1, pH 7, and the appropriate concentration of NaCI, as specified. It was purified either according to conventional methods involving ammonium sulfate precipitation, isopycnic CsCl centrifugation and gel filtration [lo], or by affinity chromatography [9]. The molecular forms were separated by sucrose gradient sedimentation ( 5 - 20% w/v sucrose in 1 M saline buffer, 1-ml sample on each gradient, centrifuged in a Spinco SW 27 rotor at 26500 rev./min, 2 "C for 32 h), and in the case of the globular forms, G, G' and G", by molecular sieve chromatography on Biogel A 1.5 m. Protein concentra- tion was determined after the method of Lowry, or by measuring the 280 nin absorption, using an absorp- tion coefficient = 16.7 [9].

Gel Filtration

Gel filtration was performed in a Biogel A 1.5 m, Biogel A 15 m or Biogel A 50 m column, as described previously [3]. The columns were equilibrated with 1 M saline buffer for acetylcholinesterase and standard proteins, except myosin. Rabbit white-muscle myo- sin, a gift from C. Klotz and B. Swynghedauw (DC- partement de Physiologie, Faculte de Medecine, Paris) was chromatographed in 0.6 M KCI, 0.01 M sodium phosphate, pH 7,l mM dithiothreitol. Phage 4, used as exclusion marker, was a gift from J. Leautey and E. Brody (Service de Biochimie, Institut de Biologie Physico-chimique, Paris). The elution parameter, KD, is equal to ( V - @/( v-- where V, v, are respec- tively the elution volume, the exclusion volume and the total volume (ferricyanide elution) [3].

Each molecular form of acetylcholinesterase was usually chromatographed separately, and its integrity was tested by sucrose gradient sedimentation after elution.

Partial Specific Volume Determination

Determination of V was obtained by a direct microdensitometric method [I11 at the Centre de Biochimie et de Biologie Moleculaire (Marseille). The density p of a solution of acetylcholinesterase (2 mg/ml) (form G) dialysed against a 0.2 M saline buffer, to- gether with the density of the buffer po, was deter- mined in a Jouan microdensitometer, at 20 "C.

po = 1.01099 k 0.00001 g/ml p = 1.011545 .t 0.00001 g/ml.

The protein concentration c was obtained from the displacement of interference fringes at a solution- buffer boundary in a centrifuge cell, using the Beck- man interferometric optical system, and assuming that dnldc = 0.4 [12], n being the refractive index of

the solution: c = 0.001997 g/ml. 5 was obtained from p = po + (1 - Vpo). The experimental errors on p and po are negligible compared to that on c. Assuming an experimental inaccuracy of 2.5% in the value for c, we obtained V = 0.714 k 0.007 ml/g, with 1% error. The drzidc factor may be the source of an additional systematic error.

Molecular Weight Determination by the Yphantis Method

Molecular weights of the four most abundant forms of acetylcholinesterase, D, C , A and G were determined by the sedimentation-diffusion equili- brium technique described by Yphantis [13].

The experiments were performed in a Beckman Spinco model E ultracentrifuge, equipped with an RTCI temperature control unit, and a laboratory- built split-beam scanner for absorption measurements [14]. Centrifugations of the G form were done in an AnD rotor, at 4 "C, 12000 rev./min using the ab- sorption optics at 280 nm. Double-sector charcoal- filled epon centerpieces, with a 12-mm pathlength, were used. The sample sector contained 50 p1 per- fluorotributylamine (FC 43) and 100 p1 of acetyl- cholinesterase solution in 0.2 M saline buffer (ab- sorbance at 280 nm was about 0.1, corresponding to a concentration of about 0.06 mgiml). The reference sector contained 20 pl perfluorotributylamine and 150 pl buffer. Scannings were performed every few hours until equilibrium was reached.

In the case of the D, C and A forms, the Rayleigh interferometric system of the centrifuge was used. Centrifugations were performed at 3 "C, in a six- channel charcoal-filled epon centerpiece. The sample channels contained 20 pl perfluorotributylamine and 100 pl solution of enzyme in 1 M saline buffer, and the reference channels were entirely filled with buffer. The D form was studied at 7200 rev./min, the A and C forms at 12000 rev./min. The plates were read with a Nikon microcomparator.

Sucrose gradient centrifugations were performed on the samples after molecular weight determination, and their activity was measured, to make sure that no degradation had occurred.

Carbohydrate Determinations

Gas Chromatography. Carbohydrate determina- tion by gas chromatography was performed according to the method of Chambers and Clamp [15]. The samples were dried, then methanolysed in 1.5-M- HC1-containing methanol, at 85 "C for 24 h, with mannitol as internal standard. After neutralization of the reaction mixture, they were converted into their

Page 3: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

S. Bon, M. Huet,

- - g 7 - " m a 73

a, zn

- .-

.- L

5 5

M.

D c 8 -

6 '

I I I I

525

Fig. 1. Sed.dimentation-d.dion equilibrium of ctret~lcholine.Ptrrus~. D, C and A forms: equilibrium was obtained at 7 200 rev./min for the D molecule (*), at 12000 rev./min for the C (m) and A (V) forms, the temperature was 3 "C; the interferometric optical system was used, the fringe displacement was measured in pm. G form (a): equilibrium was obtained at 12000 rev./min at 4 "C. The scanning equipment was used. Molecular weights were evaluated from the slopes of these plots

trimethylsilyl derivatives by reacting for 30 min at 20 "C with trimethylchlorosilane and hexamethyl- disilylazane in pyridine (1/1/5). The products were chromatographed in glass columns (diameter 0.32 cm, length 180 cm) packed with 3.5% SE 30 on WAW DMCS chromosorb (Hewlett Packard) in a 5750B Hewlett Packard chromatograph. The carrier gas was nitrogen, and the temperature rose from 100 "C at the rate of 1 "C/min.

Hexose, Hexosamine, and Sialic Acid Determina- tions. Hexoses were assayed with the orcinol method [16], using as standard a mixture which approximated the hexose composition, as found by gas chromatog- raphy (mannose: 65%, galactose 30%, glucose 5 %,).

Hexosamines were assayed according to Elson and Morgan [17], using as standard a l / l mixture of

galactosamine and glucosamine. Hydrolysis was done at 100 "C, for 4 h.

Sialic acids were assayed according to Warren [I 81, using N-acetylneuraminic acid as standard.

Hydrolysis times from 30 min to 1 h were found to yield identical results, therefore 40-min hydrolysis periods were used.

Amino Acid Composition

Amino acid analyses were performed in Prof. P. Jollb Laboratory (Laboratoire des ProtCines, Uni- versitt Paris V, Paris), after different hydrolysis times 18, 48, 72 h), under standard conditions [19]. The values reported in Table2 were those obtained after 18 h hydrolysis.

Page 4: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

526 Molecular Weight and Composition of Electrophorus Acetylcholinesterase

RESULTS

Determination of Molecular Weights by Sedimentation-DifJsion Equilibrium

The molecular weights of the four most abundant forms, D, C, A and G, were determined by the Yphantis sedimentation-diffusion equilibrium meth- od, using either interferometric optics or direct ab- sorption measurements with a scanning equipment (Fig. 1).

The partial specific volume U was determined for a preparation of acetylcholinesterase (form G) by a direct microdensitometric method, which yielded a value of 0.714 0.007 ml/g. This method required an assumption for the value of dnldc, used to determine the protein concentration, which introduced an un- certainty in the value of V. The molecular weights obtained are presented in Table 1.

Stokes Radii; Moleculur Weights of the Minor Forms

Another approach to molecular weight determi- nation, combining Stokes radii with sedimentation coefficients, allowed us to compare the molecular weights of the minor forms of acetylcholinesterase with the centrifugation data,

The Stokes radii of both asymmetric and globular forms of acetylcholinesterase were determined by molecular sieve chromatography, on Biogel A 50m and Biogel A 1.5 m respectively. The present results for the asymmetric forms are in good agreement with those previously obtained with Biogel A 15 m columns [3]; they are more accurate, however, since the elution parameters KD for Biogel A 50m are more satisfactorily located in the range where a linear re- lationship holds between the Stokes radius Re and I/-losKD (Fig. 2).

The elution pattern of the [3H]diisopropylfluoro- phosphate-labelled globular forms G, G’ and G” from a Biogel A 1.5 m column is shown in Fig. 3. The Stokes radii are given in Tablel, together with the sedimentation coefficients, obtained from sucrose gra- dient centrifugation in saline buffer. From these data molecular weights have been deduced, according to the method already outlined [3]: the products R,s of

Table 1. Molecular parameters for asymmetric and globular acetvl- cholinesteraseJorms Stokes radii R, were obtained from the elution parameters in Biogel A 50 m (D, C, A, and G forms), Biogel A 15 m (C and A forms) or Biogel A 1.5 m (G, G‘ and G” forms) columns. The data obtained from Biogel A 50 m or Biogel A 1.5 m columns, for instance, in the case of G were identical. The scatter in Stokes radii is 0.15 nm. Apparent sedimentation coefficients saPp, were obtained from sucro- se gradient centrifugations, and were reproducible to 0.1 S. The pro- ducts R,saPp were used to determine molecular weights for the less abundant forms (G’ and G”) (see Fig.4). Independent molecular weight values, obtained by the sedimentation-diffusion equilibrium method (Fig. 5 and 6) are included for the D, C, and A and G forms. Two variants of A acetylcholinesterase were encountered ; the sedi- mentation constant tended to increase upon storage, with a con- comitant decrease in Stokes radius, their products, i. e. the molecu- lar weight, remaining constant

Molecular Stokes radius Apparent Resepp Molecular form Re sedimentation weight

constant, sapp

nm S nm . S

D 15.6 18.4 287.0 1250000 204.5 796000 C 14.4 14.2

12.4 [ 11.7 A 9.1 9.65 112‘9 1 410000 112.9

G 8.75 11.8 103 331 000 G 5.91 7.7 45.6 165000” G” 3.66 5.3 19.4 70000a

~ ~ ~ ~~~

a Values obtained from ResSpp data.

0.9 -

0.8 -

0.5

0.4 - 0.3 -

I I I I

Re (nm) 0 5 10 15 20

0.2

Fig. 2. Stokes radius determinationfor the D, A and G,forms,jrom their elution parameters KD, in a Biogel A 50 m column. Standardization of the curve was done with catalase (5.2 nm), /3-galactosidase (8.2 nm), fibrinogen (10.7 nm) and myosin (21.5 nm). The intervals represent the scatter in experimental data for 5 - 10 different experiments

Page 5: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

S. Bon, M. Huet, M. Lemonnier, F. Rieger, and J . Massoulit 521

m T N

L 0

x

>

0

0 U

1 .- .- c

.- L

I G”

0.9

0.8

0.7

0.6

0.5

I I I I 50 75 100 125 150 175

Fraction number

Fig. 3. Elution profile of’ [3H]dii~oprop~lf1uorophosphate-lahelled G, G and G’ forms in a Biogel A 1.5 nz column, und Stokes radius deter- n7ination,fi-oni such elution data ( i n w r t i . The column was standardized with bovine serum albumin (3.5 nm), alcohol dehydrogenase (4.6 nm), catalase (5.2 nm), p-galactosidase (8.2 nm) and fibrinogen (10.7 nm). Exclusion (E) and total (T) volume markers were phage T4 and sodium ferricyanide. (A) 3H radioactivity: (0) absorbance at 280 nm

the two parameters are proportional to the molecular weights, since the U value is identical for all forms (see Discussion). The Yphantis values are found to be in good agreement with the relative values of R,s products, and, by comparison, allowed us to deter- mine the molecular weights of the minor forms (Fig.4 and Table 1).

Amino Acid and Carbohydrate Composition

We determined the amino acid composition of the asymmetric D form and of the tetrameric G form of acetylcholinesterase. The results are shown in Table 2, together with data obtained by Rosenberry et al. [17], with an enzyme preparation probably equivalent to G. No significant differences appear between the D and G forms and our results agree closely with those of Rosenberry et al. [20]. The polarity index, calculated according to Capaldi and Vanderkooi [21], was 47. No hydroxyproline was detectable in the chroniato- grams ; however, from preliminary results obtained with a direct assay there seems to be a small amount of hydroxyproline in the asymmetric forms, but not in form G.

The carbohydrate residues of a non-fractionated preparation of acetylcholinesterase were analyzed by

gas chromatography (Fig. 5) : neutral hexoses contain 213 mannose and 113 galactose, and hexosamines seem to contain equal amounts of N-acetylglucos- amine, and N-acetylgalactosamine ; however, one of the characteristic peaks of this sugar did not have the expected position in the chromatogram. Several small unidentified peaks appear in the fatty acid region and also a shoulder on the mannose peak. From such chromatograms we computed a 13.8% k 4% carbo- hydrate content for acetylcholinesterase.

Direct determinations yielded values of 9.25‘j/, for neutral hexoses, 3.9% for hexosamines, and 0.5% for sialic acid, giving an overall carbohydrate content of 13.65”/,, in good agreement with the above value.

DISCUSSION

The molecular weight data we report here were obtained with the Yphantis sedimentation diffusion method on one hand, and an indirect method, which involves Stokes radius and sedimentation coefficient determinations, on the other. The first method yields absolute values, but was only practicable for the most abundant forms of acetylcholinesterase. The second method, although it is based on very reproducibly

Page 6: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

528 Molecular Weight and Composition of Electrophorus Acetylcholinesterase

"0 100 200 300 Re s (nm . S)

Fig.4. Molecular weight detwmination of' the minor,fnrms cfacetyl- cholinesteruse. In this series of equal partial specific volume mole- cules, molecular weights are proportional to the product of Stokes radius by sedimentation coefficients (abscissae). Ordinates repre- sent the sedimentation-diffusion equilibrium values for D, C , A and G. The linear relationship allows determination of molecular weights for G' and G '

Table2. Amino acid composition of usyrnrnetric ( D ) and globular ( G ) acetylcholinesterase Values (mo1/100 mol) are calculated for amino acids recovered after total hydrolysis (tryptophan excluded). The data obtained in this work correspond to 38-h hydrolysis times, and those in Rosen- berry eta/ . [20] to 24 h hydrolysis. The data for D are the mean of 9 determinations, and those for G, of 2 determinations. The polarity index [21] was respectively 47.9, 46.6 and 46.8.

Amino acid Composition of acetylcholinesterase form

D G G from [17]

mo1/100 mol

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Methionine Valine lsoleucine Leucine Tyrosine Phenylabanine

5.6 2.2 4.7

31.3 5.3 7.6

11.2 6.4 9.9 7.0 2.1 2.0 6.3 4.2 8.0 2.4 4.0

6.9 2.2 3.2 9.7 4.8 7.1

12.7 4.6

11.3 7.1

1.3 6.4 3.7 7.1 2.6 4.9

-

4.6 2.3 5.2

13.1 4.5 6.8

10.3 5.9 8.7 6.2 1.6 2.7 7.0 3.x 8.6 3.6 5.3

determined data, is best used for comparative deter- minations.

The main cause of inaccuracy in molecular weight determination was due to the partial specific volume 5.

1 w L 0 a v)

L

c 0 U c

d

3 n

I\ 11

56 60 64 68 72 76 80 84 88 Time (min)

Fig. 5. Gas-liquid chromatography unalysis of rnono.pucchuride re- sidues in ace~,ylcholinr.stera.re. The peaks were identified as follows : mannose: 3, 5; galactose: 4, 6, 7; mannitol (internal standard): 11, 13; N-acetylglucosamine: 14, 16, 17. The following identifica- tions were doubtful: glucose: 8, 10 (the second peak being relatively too high); N-acetylgalactosamine: 15 (another peak was missing). Peaks 1, 2, 9 and 12 were not identified. N-acetylneuraminic acid was present but the corresponding section of the chromatogram is not shown

In a previous work 131 we estimated 2, from buoyant density measurements in cesium chloride gradients ; correct5 values cannot, however, be derived from such densities because of preferential solvation effects ; differences in buoyant densities obtained in cesium chloride us cesium sulfate clearly demonstrate such effects. It was concluded, however, that since all forms of acetylcholinesterase equilibrate at the same den- sity in a cesium chloride gradient, their 3 must be identical. This justifies the assumption of a direct proportionality between R,s products and molecular weights for all forms. The data thus obtained for the six different forms are therefore accurate at least within a common proportionality factor, and directly com- parable.

From the data shown in Tablel, it is clear that G", G' and G respectively correspond to a single monomer, a dimer, and a tetramer of the basic cata- lytic subunit. The molecular weights of D, C and A are consistent with the model we had proposed on a preliminary basis. The differences between D and C, C and A are quite compatible with the detachment of one G unit at each step. It is possible to obtain a molecular weight estimation for the common cata- lytic subunit, according to this model, from the data presented here (Fig. 6). This value, 80000, is lower than that derived from polyacrylamide gel electro- phoresis (90000; Bon and MassouliC, unpublished results). However, apart from the possibility of a systematic error in molecular weight determinations, due tov, the subunit value is likely to be biased because

Page 7: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

S. Bon, M. Huet, M. Lemonnier, F. Rieger, and J . Massoulie 529

O O F I ' 5 I I I I I 10 ' I I I I 15 ' a I

Catalytic subunit number

Fig. 6. Graphical evaluation oftlie mas.v of fhe tail,jor the asTmmetric fbrms. Molecular weights of all forms are plotted in ordinates as a function of the number of active subunits, as established in our model. The data for asymmetric and globular forms fall on two parallel straight lines. The difference is taken as an estimate for the mass of the tail

of the carbohydrate content of the polypeptides, which we have found to be close to 15%. These two inde- pendent results are therefore satisfactorily consistent.

The Problem of tlzr Tail Subunits. Amino Acid Composition

The tail is a very obvious feature in the structure of the A, C and D forms, as observed in micrographs [5,6] and perfectly accounts for their hydrodynamic properties [3,4]. Its mass may be estimated in two ways as seen in the micrographs, it is a filament about 50 nm long and 20 nm thick. In the best micrographs of the D enzyme, it seems to consist of three inter- twined strands, each attached to one tetramer. Such chains would be in the 40000-dalton range [6 ] .

We may also compute the mass of the tail if we assume that it is identical in A, C and D. Then the differences between the molecular weights of each of these forms and that of their globular heads (i. e. one, two and three monomers) represent the mass of the tail. Some part of the filament may well be included in the derived tetramers during the sequential disso- ciation, so that this difference may in fact be an under- estimate. From the figures in Table1 we obtain a value of I00000 daltons (see Fig.6). If the tail is in- deed a three-stranded structure, this implies 30000- dalton strands, which is in fair agreement with the microscopic estimate.

A three-stranded filament suggests a collagen-like structure, and indeed Taylor el al. have presented evidence that the tail of Torpedo caltjornica acetyl- cholinesterase contains collagen-characteristic amino- acids and is very sensitive to pure collagenase [22].

These authors suggest that the tail is included in the basement membrane.

If, on the other hand, the tail anchors the enzyme into the plasmic membrane, in a manner analogous to the hydrophobic peptides found in cytochrome b, [23], cytochrome b5 reductase [24] or intestinal brush border enzymes, such as aminopeptidase or sucrase- isomaltase [25,26], it may be rich in hydrophobic amino acids. No significant difference in the overall composition of asymmetric uersus globular forms of acetylcholinesterase, however, appeared. This was in fact to be expected ; although the hydrophobic peptides which may be split from detergent-solubilized amino- peptidase or maltase by trypsin were soluble in chloro- form/methanol mixtures, their amino acid composi- tions were not extremely biased in favour of hydro- phobic amino acid residues [26], and an attempt to differentiate the overall composition of the intact and trypsin-treated forms of maltase has proved unsuccess- ful [25]. Whether it is a collagen-like component of the basement membrane, or a hydrophobic membrane- bound anchor, it seems obvious that the tail somehow plays a well-defined structural role in relation to the localisation of acetylcholinesterase.

This work wa5 supported by the Ceritrr National de la Recherche Scientifiyue (Grants no. 996052 and 1858), the DPlt-gution Gt-iz6rale 2 la Recherche Srientifique et Techniyuc (Grants no. 75-7-0042 and 74-7-0368) and the Institur Nutional de la Recherche Medicale (Grant no. 74-1218-02). We thank Prof. R. Bourillon, Dr R. Cohen, Prof. P. Jolles, D r M. Charles and P. Sauve for their help, as well as Dr J . J. Bourgarit who took an active part in the preliminary stages of this work.

REFERENCES

1. Massoulie, J. & Rieger, F. (1969) Eur. J . Biochem. 11,441 -455. 2. MassouliB, J., Rieger, F. & Tsuji, S. (1970) Eur. J . Biochrm. 14,

3. MassouliC, J., Rieger, F. & Bon, S. (1971) Eur. J . Biochem. 21,

4. Bon, S., Rieger, F. & Massoulie. J . (1973) Eur. J . Biochem. 3.7,

5. Rieger, F., Bon, S., Massoulit, J . & Cartaud, J. (1973) Eur. J .

6. Cartaud. J., Rieger, F., Bon, S. & Massoulie. J . (1975) Bruin

7. €3011, S. & Massoulie, J. (1976) FEBS Lett. in press. 8. MassouliC, J., Rieger. F. & Bon, S. (1970) C. R. Hrhd. Seanc.e.s

Acad. Sci. Ser. D, Sci. Nut. (Paris), 270, 1837-1840, 9. Massoulie, J . & Bon, S. (1976) Lw. J . Biochem. 68, 531 -539.

10. Rieger, F., Bon, S., Massoulie, J., Cartaud, .I., Picard, B. &

11. Charles, M., Astier, M., Sauve, P. & Desnuelle, P. (1975) Eur.

12. Babul, J. & Stellwagen, E. (1969) Ann. Biochem. 28, 216-221. 13. Yphantis, D. A. (1964) Biochrmistry3, 297-317. 14. Cohen, R., Cluzel, J., Cohen, H., Male, P., Moignier-, M . &

Soulie, C. (1975) Bi0ph.w. C'hem. (1976) in the press. IS. Chambers, R. E. &Clamp, J. R. (1971) Biochen?. J . 125. 1000 ~

1018.

430- 439.

542-551.

372 - 379.

Biochem. 34, 539- 547.

Res. 88, 127- 130.

Benda, P. (1976) Eur. J . Biochem. 68, 513-521.

J . Biochem. 58, 555 - 559.

Page 8: Molecular Forms of Electrophorus Acetylcholinesterase : Molecular Weight and Composition

530 S. Bon et a/. : Molecular Weight and Composition of Electrophorus Acetylcholinesterase

36. Lustig, B. & Langer, A. (1931) Biochem. 2. 242, 320-337. 17. Elson, L. A. & Morgan, W. T. (1953) Biochem. J . 27, 1824-

18. Warren, L. (1954) J . Biol. Chem. 234, 1971 -1976. 19. Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal.

20. Rosenberry, T. L., Chang, H. W. & Chen, Y. T. (1972) J . Bid.

21. Capaldi, R. A . & Vanderkooi, G. (1972) Proc. Natl Acad. Sci.

1828.

Chem. 30,1190-1206.

Chem. 247, 1555-1565.

U.S.A. 69,930 - 932.

22. Taylor, P., Jones, J. W. & Jacobs, N. M. (1974) Mot. P lwwm-

23. Spatz, L. & Strittmatter, P. (1971) Proc. Nut1 Acad. Sci. U.S.A.

24. Spatz, L. & Strittmatter, P. (1973) J . B id . Chem. 248,793 -799. 25. Sigrist, H., Ronner, P. & Semenza, G. (1975) Biochim. Biophys.

26. Maroux, S . & Louvard, D. (1976) Biochim. Biophys. Acta, 419,

col. I0,93 - 107.

68,1042 - 1046.

Acta, 406, 433 -446.

189-195.

S. Bon, F. Rieger and J. Massoulit, Laboratoire de Neurobiologie, Ecole Normale Superieure, 46 Rue d’Ulm, F-75230 Paris-Cedex-05, France

M. Huet, Laboratoire de Physique Biologique. Institut de Biologie Moleculaire, Universite Paris VII, 2 Place Jussieu, F-75221 Paris-Cedex-05, France

M. Lemonnier, Centre de Recherches snr les ProtCines, Faculte de MCdecine Lariboisiere-Saint-Louis, 45 Rue des Saints-Ptres, F-75270 Paris-Cedex-Oh, France