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Molecular forms of acetylcholinesterase in developing Torpedo embryos
Nearochemistry International,Vol. 4, No. 6, pp. 577 to 585, 1982. Printed in Great Britain.
0197-0186/82/060577-09503.00/0 © 1982 Pergamon Press Ltd.
MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN DEVELOPING TORPEDO EMBRYOS SUZANNE BON Laboratoire de Neurobiologie, Ecole Normale Sup6rieure, 46, rue d'Ulm 75230 Paris Cedcx 05, France
(Received 20 April 1982; accepted 3 June 1982) Abstract--We have studied the evolution of acetylcholinesterase molecular forms during the embryonic devel-
opment of Torpedo rnarrnorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, 'myogenic phase' of electric organ development), globular forms of acetylcholinesterase (G4 and G2) are abundant in both tissues and the collagen-tailed form A12 is already present. In the electric organs, this form accumulates rapidly after the 55-60mm stage ('eleetrogenic phase'), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCI2, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form As shows a transient increase which coincides approximately with the maximal accumulation of At2, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G2, which eventually becomes predominent in the adult electric organs, lags behind that of A12. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of At2. In the electric lobes, the tetrameric form (G4) is abundant during development, as well as G2 and G~ at certain stages, but the A~2 form is predominant in the adult.
The electric organs of Electrophorus and Torpedo pro- non catalytic subunits appear to replace some of the vide an exceptionally favourable material for the bio- catalytic subunits (Lee, Camp and Taylor, 1982), we chemical study of proteins involved in cholinergic use the same terminology, because it conveniently transmission. We have previously shown that acetyl- expresses the homology which exists between the cholinesterase (EC 3.1.1.7) occurs in several distinct enzyme forms of the two species. molecular forms in these organs (Bon, Huet, LemonIn addition to the collagen-tailed forms, Torpedo nier, Rieger and Massouli6, 1976; Bon and Massouli~, electric organs contain a major proportion of hydro1980). phobic acetylcholinesterase dimers, which form comElectrophorus electric organs Contain almost exclus- plexes with non-denaturing detergents such as Triton ively collagen-tailed forms, in which catalytic X100 (Bon and Massouli6, 1980; Viratelle and Berntetramers are attached to a collagen-like element. We hard, 1980; Witzemann, 1980), sedimenting at about have called these asymmetric forms A4, As and A,2, 6S. A partial digestion of the 6S (G2) hydrophobic according to their number of catalytic subunits (Bon enzyme by pronase or proteinase K produces active et al., 1976; Bon, Vigny and Massouli6, 1979). These dimers which no longer interact with Triton X100, and thereby resemble those obtained by cleavage of molecules possess characteristic aggregating properthe collagen-tailed forms (Bon and Massouli6, 1980). ties in the presence of acidic polysaccharides (Bon, Cartaud and Massouli6, 1978), indicating ionic interThe hydrophobic character of the 6S acetylcholinactions which are thought to anchor acetyicholin- esterase is therefore due to a hydrophobic peptide or domain which may be removed or disorganized by esterase in situ within the extracellular basal lamina proteolysis. We have proposed that the hydrophobic (Lwebuga-Mukasa, Lappi and Taylor, 1976). Torpedo electric organs also contain collagen-tailed acetyl- enzyme might be a biosynthetic precursor of the collagen-tailed forms. In this case the'collagen-tailed cholinesterase, mostly two formswhich resemble the Electrophorus A~2 and As forms in their physico-che- forms would correspond to a mature state of the mical properties (Bon and Massouli6, 1980). Although enzyme and perhaps represent its physiologically the quaternary structure of these molecules is in fact active fraction, as in Electrophorus where they constidifferent from that of the Electrophorus forms, since tute the quasi-totality of acetylcholinesterase. 577 N.C.L4/6--t
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In the skeletal muscles of birds and mammals, the collagen-tailed forms of acetylcholmesterase are controlled in a specific manner by inner~ation, as shown by their appearance during embryogenesis and by their disappearance after denervation (Massoulid, Bon and Vigny, 1980; Massouli6 and Bon. 1982). It would be interesting to know whether such a neural control also exists in the case of a primitive vertebrate such as Torpedo. Denervation of the electric organs, which represent modified muscles, may not answer this question readily because of the stow rate of nerve degeneration in this species. It was therefore of particular interest to examine whether (1) the different forms appeared in a determined sequence during the embryonic development of Torpedo and (2) a correlation exists between their biosynthesis and synapse formation. Torpedoes are oviviviparous, so that embryos must be obtained from pregnant females. The developmental stages of the embryos have been well described from the morphological and biochemical points of view (Mellinger, Belbenoit, Ravaille and Szabo, 1978: Fox and Richardson, 1978, 1979: Krenz, Tashiro, W~ichtler, Whittaker and Witzemann, 1980: Richardson, 1980), with special emphasis on the electric organ and its innervation by the motoneurons of the electric lobe, the brain region which c o m m a n d s the electric discharge. The present study described the distribution of acetylcholinesterase molecular forms in the electric organs and in the electric lobes, from an early stage until birth.
I)ation~, of MgC'I> The mixtures ~%c)-eincubah:d ,*; 20 ( iZ)r 30 rain, centrifuged (Sorvall SS 34, 15 rain at 15. )00 rex mini and the supernatants were assayed l'oJ accl~lcholineslerase activity. Molecular forms of acetylcholinestcrase x~crc amd~zcd by centrifuging an aliquot of the total tissue extract in a sucrose gradient (5 20°0 w/v sucrose, in I M NaCL 5~) mM MgCI2, 10mM Tris HCL pH 7, 0.1 mgml bacitra~:in_ 1",, Triton XI001 in a SW41 Beckman rotor, at 4 t'. 40,000rev'min (Bon and Massouli4. 1980L Sedimentation coelficients were determined by comparison with /;-galactosidase from E. coil (16 S), catalase from beef liver (I 1.3 S] and alkaline phosphatase from calf intestine 16,1 S}. included in the gradients as internal standards. The hydrolysis of acetylthiocholine was assayed by the spectrophotometric method of Ellman, Courtney, Andres and Featherstone (1961) at 28 C, and the actMties are expressed as o.d units per gram of fresh tissue (1 o.d unit corresponds to 75 nmol of acetylthiocholine hydrolyzedL It should be noted that the relative values of the activities obtained at different stages and the proportions of the molecular [orms are independent of the assay conditions, although these may influence the absolute values. We have xerilicd, by their sensitivity to the specific inhibitor BW 284 C 5t,* that all molecular forms correspond to true acetylcholii~csterase (EC 3.1.1.7.1. R ESLJLTS (A) Proportions q/ acety/cholinesterase molecu/ar.lorms
in developin¢] electric oryan.s and electric lobes o1 Torpedo Acetylcholinesterase is present in the electric organs and in the electric lobes of Torpedo embryos as early ' I
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EXPERIMENTAL PROCEDURES
Torpedo embryos were obtained from pregnant females in summer at the Station de Biologie Marine of Arcaehon (France). Different developmental stages were found at different times, and characterized by the body length of the embryos (Mellinger et al., 1978: Krenz et al., 1980). The electric organs and electric lobes were immediately dissected, and samples were kept frozen at - 80 C. until use. Acetylcholinesterase was quantitatively solubilized by homogenizing the tissues with 5 volumes of high salt buffer, in the presence of detergent (1 M MgCI2, 0.01 M Tris HC1, pH7. 1!~,~ Triton XI00, 0.1 mg/ml baeitracin) (Bon and Massoulie, 19801. We prepared suspensions of particulate collagen-tailed forms in two different manners: (a) the tissues were homogenized in 10 volumes of low salt buffer (10raM Tris HCI, pH 7), (b} the supernatant of a high salt extraction (1 mM MgCI> 10mM Tris HC1, pH 7) was dialyzed against the low salt buffer. These suspensions were then diluted with 20 volumes of 10mM Tris HC1 pH 7 buffer, containing 0.1 mg/ml bacitracin, and various concen* l, 5- Bis(4- allyldimethylammoniumphenylt- pentane- 3 one dibromide (Sigma}.
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Fig. 1. Variations of acetylcholinesterase activity in the electric organs and electric lobes of Torpedo during embryonic development. The activity per gram of fresh tissue, determined by the method of Ellman et at. (196l) is plotted as a function of body length, electric lobes: electric organs: our data (@) and the values given by Krenz et al. (1980) (O) have been normalized for 80ram. The variations at early stages are shown with a larger scale in the inset.
Acetylcholinesterase forms in Torpedo embryos as the 35 mm body length stage. As shown in Fig. 1, the level of the enzyme remains low and approximately equal in both tissues until the 50 mm stage, when it reaches about 1% of the value observed at birth in the electric organs. As already reported by Krenz et al. (1980), acetylcholinesterase then increases rapidly, particularly in the electric organs, which after the 60 mm stage contain a much greater activity than the electric lobes. Sedimentation patterns of acetylcholinesterase, corresponding to different stages of development are illustrated in Fig. 2: they show that the increase in the enzyme activity is accompanied by complex variations of its composition in terms of molecular forms. The profiles given in Fig. 2 demonstrate that besides the A12 (17.5 S), Aa (13.8 S) and hydrophobic G 2 (6 S) forms, the tissues contain two molecular forms, sedimenting at 3.6S and 10.8 S, which have not been identified in detergent extracts of adult electric organs. These forms exist in the nervous tissue of adult Torpedo and appear to represent hydrophobic monomers (G1) and tetramers (G4) (Bon and Toutant, in preparation). The smaller A4 form is never abundant, but is detectable at the 50-60ram stages; it is clearly recognized in an aggregating fraction prepared from an extract of 58 mm electric organ (Fig. 3). From the sedimentation patterns of acetylcholinesterase, it is possible to estimate the contribution of each molecular form to the total activity. Figure 4 summarizes the variations of these proportions in the electric organs. The experimental points, representing the values obtained with different embryos, show a scatter of about 10%, because of experimental errors due to the incomplete resolution of the sedimentation patterns and probably also because of individual differences in the development of the embryos. The relative variations of the different forms are however quite clear. In the early stages of electric organ development, the hydrophobic G 2 and G4 forms are predominant, but A12 is already present; A12 then increases in proportion and is accompanied by As which transiently represents the most abundant form during the 50-60mm stages. G2 increases later: G2 and A12 are essentially the only forms present at the time of birth, G 2 being more abundant than A12. In the adult, A8 becomes visible again. By combining the relative proportions given in Fig. 4 with the evolution of the total activity in the electric organ, it is possible to evaluate approximately the absolute activity of each form during development. We have multiplied this value by the weight of the electric organ, obtained from Mellinger et al. (1978), and the results are given in Fig. 5, as a function of
579
body length. Considering that the growth of the embryos is approximately constant (1 mm/day) between the 30 and l l 0 m m stages (Krenz et al., 1980), these curves provide a rough approximation of the rate of accumulation of the different forms in the electric organs. They show that the activity increases markedly after the 60°70 mm stage, corresponding to an accumulation of A12, later followed and overtaken by an accumulation of G2. We did not construct a similar set of curves in the case of the electric lobes, which we have not investigated as extensively. As in the electric organs, Ga occurs together with G 2 and A12 at early stages but later decreases continually; A12 rapidly increases, becoming predominant around the 50mm stage. Its proportion transiently decreases, around the 800100 mm stages, as the smaller globular forms G1 and G 2 become relatively abundant (about 20°/0 of the total activity). The adult lobes contain a major proportion of A12, together with a small proportion of As. It is remarkable that As is not observed in the embryonic stages, but appears in the adult. (B) Solubilization and aooreoation properties of collagen.tailed forms in embryonic and adult Torpedo The analyses described in the previous section were performed with tissue extracts prepared in a high salt, detergent-containing medium, in order to solubilize quantitatively all forms of acetylcholinesterase. We also examined the influence of salt concentration on the solubility of the collagen-tailed forms in electric organs and electric lobes, hoping that it might reveal possible differences in their attachment to basal lamina or other structures. When the tissues were homogenized in 10mM Tris-HC1 pH 7, in the absence of divalent cations, a fraction of collagen-tailed acetylcholinesterase was recovered in the supernatant. This fraction was notably higher after a low speed centrifugation (Sorvall SS 34, 13,000rev/min, corresponding to 15,0009, 15min) than after a high speed centrifugation (Airfuge, maximal speed - 95,000 rev/min, corresponding to ~100,000g, 5min): we obtained respectively about 50 and 20% of the total activity in the case of the electric lobe. We found by sedimentation analysis in low salt sucrose gradients that in such electric lobes supernatants the collagen-tailed enzyme does not occur as individual molecules, but as potydisperse aggregates (around 50-70 S) explaining the variability of the yield of this low salt 'extraction'. In any case, it is clear that the fraction of these forms which is thus extracted in low salt is much smaller in the case of electric organs (5-10%) than in electric lobes.
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Fig. 3. Presence of the smallest asymmetric form (A+) in aggregates from a 58 mm embryo electric organ extract. A high salt extract of 58 mm embryo electric organ was fractionated into soluble and aggregated components after dialysis against a low salt buffer containing 40 mM MgCI2. The precipitated material was redissolved in high salt buffer prior to centrifugation in sucrose gradient (cfi Fig. 2). O O the soluble components correspond to the G+ and G 2 globular forms, g --Q the aggregated components correspond to the three asymmetric forms A12. As and clearly A,. The collagen-tailed molecules precipitate in the presence of a low concentration of divalent cations, and become solubilized by high concentrations of MgCIz (Fig. 6A). We compared the influence of MgCi2 concentration in two different experiment~il conditions: the solubilization of acetylcholinesterase from the particulate fraction of a tissue homogenate which had been prepared in low salt, and the dissociation of aggregates which had been precipitated by dialysis of a high salt solubilized extract against a low salt buffer. In the case of the electric lobes, we obtained essentially the same curve for the low speed 'soluble' and 'insoluble' fractions resulting from homogenization of the tissue in 10 mM Tris-HCl buffer, as well as for high salt solubilized, reaggregated enzyme. The dissociation of reaggregated collagen-
tailed forms from electric organ was achieved at a much smaller MgC12 concentration, than that required from their dissociation from the tissue homogenate indicating that interactions in the native structures differ from those which occur in aggregates obtained in vitro. The solubilization curves of the electric organ enzyme (but not the dissociation of reaggregated enzyme) in fact depends upon the developmental stage: Fig. 6(B) shows that the collagen-tailed enzyme becomes more difficult to solubilize as the electrocytes mature. It must finally be noted that the collagentailed forms possess identical sedimentation coefficients, and show their characteristic sensitivity to collagenase at all developmental stages, suggesting that they have the same molecular structure.
Fig. 2 (opposite). Sedimentation patterns of acetylcholinesterase at various developmental stages. (A)-(D): electric organs; (E)-(H): electric lobes. Tissue extracts were centrifuged in sucrose gradients (see "Experimental Proc6dures"). The acetylcholinesterase activity is plotted on an arbitrary scale, as a function of the fraction number. Migration is from the right to the left. The curves have been normalized to the same maximal value, and the corresponding forms brought into coincidence. The positions of the sedimentation standards //-galactosidase (16 S), catalase (11.3 S) and alkaline phosphatase (6.1 S) are indicated by arrows in frames A and E. The embryo length is indicated in the upper right corner of each graph.
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Fig. 4. Variations of the proportions of acetylcholinesterase molecular forms during development of Torpedo electric organ. Proportions of the molecular forms were estimated from the sedimentation patterns (of Fig. 21. • " • G2 101; --G4 (•); A8 (A) and Al2 (I/.
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DISCUSSION In this study, we analyzed the molecular forms of acetylcholinesterase from Torpedo embryonic electric organs and electric lobes homogenized in a medium containing 1 M MgClz and 1°41 Triton XI00, which allows the complete solubilization of b o t h collagen-
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Fig. 6. (A) Influence of salt concentration on the solubilization of collagen+tailed forms in adult electric organ and electric lobe. The full symbols correspond to the solubilization of collagen-tailed acetylcholinesterase, obtained by incubating a particulate suspension prepared by homogenizing the tissue in a low salt burlier (10mM Tris HCI, pH 7). The empty symbols correspond to the dissociation of aggregates obtained by dialysis of high salt solubitized enzyme against the low salt buffer. • + 0 electric organ: A, A electric lobe. The activities recovered in the supernatants Isee "'Experimental Procedures") (as compared to the total collagen-tailed acetylcholinesterase) are plotted as a function of the final MgCI2 concentration. (B) Solubitization of collagen-tailed acetylcholinesterase forms from low-salt homogenates of electric organs at various embryonic stages. ~ 41 mm: A 58mm: • 70mm and • adult.
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Fig. 5. Evolution of the total activity of acetylcholincsterase molecular forms in Torpedo electric organs, during embryonic development. "'- G2:--- G 4 : A~ and A~z. The activity per gram of electric organ was evaluated by applying the proportions determined in Fig. 2 to the total activity (Fig. 1). The result was multiplied by the weight of the electric organ, obtained from the curves given by Mellinger et al. (1978). The evolutions of the minor G 4 and A~ forms arc shown with a larger scale in the inset.
tailed and globular forms of the enzyme, including its hydrophobic fraction. We thus observed the existence of m o n o m e r i c and tetrameric hydrophobic acetylcholinesterase which had not yet been identified as native forms in detergent extracts of adult electric organs. We recently demonstrated that in addition to the 6 S dimeric form, nervous tissues from adult Torpedo contain collagenase-insensitive hydrophobic forms of acetylcholinesterase, which correspond to monomeric (3.6S) and tetrameric (9.8S) molecules (Bon and Toutant, in preparation). Torpedo tissues therefore contain three globular forms, homologous to the G~, G 2 and G+ forms observed in higher vertebrates (Bon et al., 1979; Grassi, Vigny and Massouli6, 1982).
Acetylcholinesterase forms in Torpedo embryos One aim of this study was to examine whether the different forms appear in a definite sequence, which might correspond to progressive stages in their biosynthesis. If this were the case, G1 and G2 would have been expected to precede G4. On the contrary, G4 is found as a major component of acetylcholinesterase at early stages (before the 50 mm stage) but then decreases steadily, in both the electric organs and the electric lobes. In the electric organs, G~ has not been identified at any stage, and hydrophobic G 2 passes through a minimum, in relative terms, in 60-70 mm stage embryos. In the electric lobes, the proportion of G1 passes through a maximum, around the 80-100mm stage. Clearly these variations cannot be simply related to biosynthetic relationships between the three globular forms. In a similar manner, it is not possible to consider the hydrophobic G2 form, which represents a high proportion of enzyme in the adult electric organs, as a pool of precursors of the collagen-tailed forms, since A~2 accumulates earlier than G2. It is not possible at the moment to decide whether distinct genes direct the synthesis of globular and collagen-tailed acetylcholinesterase, or whether the assembly lines leading to the two types of molecules are organized in such a way that biosynthetic intermediates do not normally accumulate in a conspicuous manner. As for the collagen-tailed forms, the most salient feature is probably the transient appearance of A4 and A8 forms in the electric organs about the 55 mm stage. As indicated in Fig. 4, the highest specific activity of the Aa form corresponds to the onset of the rapid increase of A12 (60-100mm). This of course suggests that As might represent an intermediate in the assembly of At2, and that its relatively high level would be directly correlated with an active synthesis of the larger collagen-tailed form. The fact that As was not observed in the electric lobes during the embryonic stages may be related to the lower rate of A~2 synthesis in the nervous tissue. It is also remarkable that although As is absent at birth, it occurs at the adult stage both in the electric organs and in the electric lobes. It may be that the A forms represent different molecular structures at different stages since it has recently been shown that catalytic subunits may be partly replaced to variable degrees by non-catalytic subunits of unknown function (Lee et al., 1982). We must now examine whether any correlations can be established between the differentiation of the electrocytes, particularly their innervation by electric lobe motoneurons, and the appearance of specific forms of acetylcholinesterase. The studies of Mellinger et al. (1978) and Fox and Richardson (1978, 1979)
583
have established that the development of the electric organ can be divided in two phases: a myogenic phase and an electrogenic phase. During the myogenic phase, i.e. until the 40 mm stage, the cells possess the characteristics of embryonic myotubes, they contain myofibrils, and are vertically oriented. Between the 40 and 55 mm stages, the cells become horizontally flattened and lose their organized myofibrils. After the 55 mm stage, the level of acetylcholine receptor increases strongly (Krenz et al., 1980). Nerve terminals may be seen in the electric organs after the 60-70 mm stages, but only extremely small electrical responses can be elicited at these early stages by stimulating the electric nerve. Electric organ discharge amplitude begins to increase exponentially after the 90 mm stage and reaches adult values at birth. Studies of acetylcholinesterase forms during the development of rat and chicken have established that globular forms exist very early in skeletal muscles but that the appearance of collagen-tailed forms is concomittant with the establishment of neuromuscular contacts (Massouli6 et al., 1980; Massouli6 and Bon, 1982). It was therefore surprising to find that the A12 form already represents a significant proportion of acetylcholinesterase in the electric organs at the earliest stage which we investigated (35 mm) i.e. before the end of the myogenic phase of development and before synapse formation begins. The collagen-tailed forms of acetylcholinesterase therefore do not appear to be controlled in the same manner in the Torpedo electric organs as in the muscles of higher vertebrates. However, it is interesting to note that the A~2 form starts to increase rapidly in absolute amount at the 55-60mm stage, i.e. when the electrocytes become morphologically differentiated and when the ingrowth of electromotor nerves into the inter electrocyte space begins. It is probably relevant that the basal lamina following the ventral face of the electrocytes is already visible at this stage (Richardson, 1980), since the collagen-tailed forms are thought to be localized in these extracellular structures. It is remarkable that the major accumulation of G2, which occurs later than that of A~2 coincides with the major development of nerve terminals and with the period of appearance and increase of electric organ discharge amplitude (Krenz et al., 1980), in agreement with recent findings which indicate that this form is largely presynaptic (Morel and Dreyfus, 1982; Li and Bon, 1982). This suggests an important physiological significance of G2 acetylcholinesterase at the presynaptic sites. It may be surprising, in this view, that G2 never reaches very high levels in the electric lobes, where A12 in fact becomes largely predominant
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in the adult, The cytochemical distribution of acetylcholinesterase activity in the cell body of the motor neurons (Tsuji, 1977) suggests that this A~ 2 enzyme is mostly intracellular, in contrast with the electric organs. The characteristics of solubilization of the collagentailed forms are indeed different in the electric organs and electric lobes, in two respects. Firstly, the proportion of collagen-tailed acetylcholinesterase which is recovered in the low speed supernatant after a low salt homogenization is markedly higher in the electric lobes, as also observed by W i t z e m a n n and Boustead (1981). We have shown that this enzyme, in fact. represents polydisperse aggregates rather than isolated molecules, so that the yield of low salt 'extracted' collagen-tailed acetylcholinesterase markedly depends upon the centrifugation conditions. Secondly, the solubilization of collagen-tailed forms of acetylcholinesterase by salt requires a higher concentration of MgCI2 in the electric organs than in the electric lobes where it followed the curve observed for dissociation of previously solubilized, in ritro reaggregated enzyme. Furthermore, the solubilization of collagentailed enzyme from electric organs occurs at increasingly high MgCI: concentrations, during the course of maturation. These variations do not appear correlated with intrinsic differences in the collagen-tailed molecules themselves, and in particular their aggregation properties appear similar, as indicated by the dissociation of in t:itro aggregated enzyme. The distinct solubilization properties of these enzymes in electric lobes and electric organs therefore probably result from their intracellular localization in the first case, and the evolution of more complex and stable associations within the developing basal lamina in the second case. In conclusion, the present study revealed that during the development of the electric organs and the electric lobes of Torpedo, the different molecular forms of acetylcholinesterase present a complex evolution which is not readily correlated with biosynthetic sequences, but rather reflects the morphological and physiological differentiation of the nervous and electric tissues.
Acknowledgements I wish to thank particularly Dr. Guy Richardson, who provided the Torpedo embryos, for his help with the dissections, and for very useful advice. I am grateful also to Dr. Jean Massouli6 for his encouragement throughout the course of the work and for helpful discussions, and to Pierre Allemand for his invaluable technical assistance. This work was supported by grants of the Centre National de la Recherche Scientifique, and the Direction Gdn6rale h la Recherche Scientifique et Technique.
REFEREN('):S Bon, S., Huet, M., Lemonnier, M., Rieger, I~. and Massouli& J, (1976L Molecular forms of l£1ectrophoru~ acclylcholinesterase. Eur, J. Biochem. 6g, 523 53/), Boil, S., Cartaud, J. and Massoulie, J. (1978!. The dependence of acetylcholinesterase aggregation at low ionic strength upon a polyanionic component. Era'. I. Biochem. 85. 1 14. Bon, S., Vigny, M. and Massoulie. J. (1979). Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. hath. Acad. Sci. U.S.A. 76, 2546 2550. Bon, S. and Massoulie. J. (19801, Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organs. Proc. hath. :lead. Sei. ('.S.A. 77, 4464 4468. Etlmann. G. L., Courtney, K. D., Andres, V. and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmac. 7, 88-95. Fox. C. Q. and Richardson, G. P. (1978). The developmental morphology of Torpedo marmorata: electric organmyogenic phase, J. comp. Neurol. 179, 677 697. Fox. G. Q. and Richardson. G. P, (19791. The developmental morphology of Torpedo marmorata: electric organelectrogenic phase. J. eomp. Nem'ol. 185, 293 314. Grassi. J., Vigny, M. and Massouli6, J. (1982), Molecular forms of acetylcholinesterase in bovine caudate nucleus and superior cervical ganglion: solubility properties and hydrophobic character. J. Neuroehem. 38, 457 469 Krcnz, W. D.. Tashiro, T,, WS.chtler. K., Whittaker. V. P. and Witzemann. V. (1980). Aspects of the chemical embryology of the etectromotor system of Torpedo marmorata with special reference to synaptogenesis, ~'euroscience 5, 617 624. Lec. S.. Camp, S. and Taylor, P. (1982i. Structural characterization of the asymmetric (17S + 13 S) species of acetylcholinesterase from Torpedo: analysis of subunit composition. 3. biol. Chem. in press. Li, Z. Y. and Bon, C. (1982). Presence of a membranebound acetylcholinesterase form in a nerve-endings preparation from Torpedo marmorata electric organ, J. Neurochem. in press. Lwcbuga-Mukasa, J. S.. Lappi, S. and Taylor, P. 11976). Molecular forms of acetylcholinesterase from Torpedo calilorniea: their relationship to synaptic membranes. Biochemi,~try 15, 1425 1234. Massouli6, J., Bon, S. and Vigny, M. (1980). The polymorphism of cholinesterases in vertebrates. Neuroehem. Int. 2. 161 184. Massouli,L J. and Bon, S, (1982). The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. Ann. Rer, Neurosci. 5, 57 106. Mellinger, J., Belbenoit. P., Ravaillc, M. and Szabo. 1-. (I978). Electric organ development in Torpedo marmorata, Chondrichthyes. Dec. Biol. 67, 167 188. Morel, N. and Dreyfus, P. (1982). Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ. Neurochemistry Int. 4, 283 288. Richardson. G. P. (19801. Development of the eleclromotor system of Torpedo nlarnun'ata. PhD, Thesis, University of Sussex. Tsuji. S. (19771. Acetylcholinesterasc positive elcctromotor
Acetylcholinesterase forms in Torpedo embryos neurons in the electric lobes of Torpedo marmorata. Brain Res. 124, 352-356. Viratelle, O. M. and Bernhard, S. A. (1980). Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated. Biochemistry 19, 4999-5007. Witzemann, V. (1980). Characterization of multiple forms
585
of acetylcholinesterase in electric organ of Torpedo marmorata. Neurosci. Lett. 20~ 277-282. Witzemann, V. and Boustead, C. (1981). Distribution of acetylcholinesterase molecular forms in brain, nerve and muscle tissue of Torpedo marmorata. Neurosci. Lett. 26, 313-318.
0197-0186/82/060577-09503.00/0 © 1982 Pergamon Press Ltd.
MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN DEVELOPING TORPEDO EMBRYOS SUZANNE BON Laboratoire de Neurobiologie, Ecole Normale Sup6rieure, 46, rue d'Ulm 75230 Paris Cedcx 05, France
(Received 20 April 1982; accepted 3 June 1982) Abstract--We have studied the evolution of acetylcholinesterase molecular forms during the embryonic devel-
opment of Torpedo rnarrnorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, 'myogenic phase' of electric organ development), globular forms of acetylcholinesterase (G4 and G2) are abundant in both tissues and the collagen-tailed form A12 is already present. In the electric organs, this form accumulates rapidly after the 55-60mm stage ('eleetrogenic phase'), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCI2, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form As shows a transient increase which coincides approximately with the maximal accumulation of At2, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G2, which eventually becomes predominent in the adult electric organs, lags behind that of A12. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of At2. In the electric lobes, the tetrameric form (G4) is abundant during development, as well as G2 and G~ at certain stages, but the A~2 form is predominant in the adult.
The electric organs of Electrophorus and Torpedo pro- non catalytic subunits appear to replace some of the vide an exceptionally favourable material for the bio- catalytic subunits (Lee, Camp and Taylor, 1982), we chemical study of proteins involved in cholinergic use the same terminology, because it conveniently transmission. We have previously shown that acetyl- expresses the homology which exists between the cholinesterase (EC 3.1.1.7) occurs in several distinct enzyme forms of the two species. molecular forms in these organs (Bon, Huet, LemonIn addition to the collagen-tailed forms, Torpedo nier, Rieger and Massouli6, 1976; Bon and Massouli~, electric organs contain a major proportion of hydro1980). phobic acetylcholinesterase dimers, which form comElectrophorus electric organs Contain almost exclus- plexes with non-denaturing detergents such as Triton ively collagen-tailed forms, in which catalytic X100 (Bon and Massouli6, 1980; Viratelle and Berntetramers are attached to a collagen-like element. We hard, 1980; Witzemann, 1980), sedimenting at about have called these asymmetric forms A4, As and A,2, 6S. A partial digestion of the 6S (G2) hydrophobic according to their number of catalytic subunits (Bon enzyme by pronase or proteinase K produces active et al., 1976; Bon, Vigny and Massouli6, 1979). These dimers which no longer interact with Triton X100, and thereby resemble those obtained by cleavage of molecules possess characteristic aggregating properthe collagen-tailed forms (Bon and Massouli6, 1980). ties in the presence of acidic polysaccharides (Bon, Cartaud and Massouli6, 1978), indicating ionic interThe hydrophobic character of the 6S acetylcholinactions which are thought to anchor acetyicholin- esterase is therefore due to a hydrophobic peptide or domain which may be removed or disorganized by esterase in situ within the extracellular basal lamina proteolysis. We have proposed that the hydrophobic (Lwebuga-Mukasa, Lappi and Taylor, 1976). Torpedo electric organs also contain collagen-tailed acetyl- enzyme might be a biosynthetic precursor of the collagen-tailed forms. In this case the'collagen-tailed cholinesterase, mostly two formswhich resemble the Electrophorus A~2 and As forms in their physico-che- forms would correspond to a mature state of the mical properties (Bon and Massouli6, 1980). Although enzyme and perhaps represent its physiologically the quaternary structure of these molecules is in fact active fraction, as in Electrophorus where they constidifferent from that of the Electrophorus forms, since tute the quasi-totality of acetylcholinesterase. 577 N.C.L4/6--t
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In the skeletal muscles of birds and mammals, the collagen-tailed forms of acetylcholmesterase are controlled in a specific manner by inner~ation, as shown by their appearance during embryogenesis and by their disappearance after denervation (Massoulid, Bon and Vigny, 1980; Massouli6 and Bon. 1982). It would be interesting to know whether such a neural control also exists in the case of a primitive vertebrate such as Torpedo. Denervation of the electric organs, which represent modified muscles, may not answer this question readily because of the stow rate of nerve degeneration in this species. It was therefore of particular interest to examine whether (1) the different forms appeared in a determined sequence during the embryonic development of Torpedo and (2) a correlation exists between their biosynthesis and synapse formation. Torpedoes are oviviviparous, so that embryos must be obtained from pregnant females. The developmental stages of the embryos have been well described from the morphological and biochemical points of view (Mellinger, Belbenoit, Ravaille and Szabo, 1978: Fox and Richardson, 1978, 1979: Krenz, Tashiro, W~ichtler, Whittaker and Witzemann, 1980: Richardson, 1980), with special emphasis on the electric organ and its innervation by the motoneurons of the electric lobe, the brain region which c o m m a n d s the electric discharge. The present study described the distribution of acetylcholinesterase molecular forms in the electric organs and in the electric lobes, from an early stage until birth.
I)ation~, of MgC'I> The mixtures ~%c)-eincubah:d ,*; 20 ( iZ)r 30 rain, centrifuged (Sorvall SS 34, 15 rain at 15. )00 rex mini and the supernatants were assayed l'oJ accl~lcholineslerase activity. Molecular forms of acetylcholinestcrase x~crc amd~zcd by centrifuging an aliquot of the total tissue extract in a sucrose gradient (5 20°0 w/v sucrose, in I M NaCL 5~) mM MgCI2, 10mM Tris HCL pH 7, 0.1 mgml bacitra~:in_ 1",, Triton XI001 in a SW41 Beckman rotor, at 4 t'. 40,000rev'min (Bon and Massouli4. 1980L Sedimentation coelficients were determined by comparison with /;-galactosidase from E. coil (16 S), catalase from beef liver (I 1.3 S] and alkaline phosphatase from calf intestine 16,1 S}. included in the gradients as internal standards. The hydrolysis of acetylthiocholine was assayed by the spectrophotometric method of Ellman, Courtney, Andres and Featherstone (1961) at 28 C, and the actMties are expressed as o.d units per gram of fresh tissue (1 o.d unit corresponds to 75 nmol of acetylthiocholine hydrolyzedL It should be noted that the relative values of the activities obtained at different stages and the proportions of the molecular [orms are independent of the assay conditions, although these may influence the absolute values. We have xerilicd, by their sensitivity to the specific inhibitor BW 284 C 5t,* that all molecular forms correspond to true acetylcholii~csterase (EC 3.1.1.7.1. R ESLJLTS (A) Proportions q/ acety/cholinesterase molecu/ar.lorms
in developin¢] electric oryan.s and electric lobes o1 Torpedo Acetylcholinesterase is present in the electric organs and in the electric lobes of Torpedo embryos as early ' I
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EXPERIMENTAL PROCEDURES
Torpedo embryos were obtained from pregnant females in summer at the Station de Biologie Marine of Arcaehon (France). Different developmental stages were found at different times, and characterized by the body length of the embryos (Mellinger et al., 1978: Krenz et al., 1980). The electric organs and electric lobes were immediately dissected, and samples were kept frozen at - 80 C. until use. Acetylcholinesterase was quantitatively solubilized by homogenizing the tissues with 5 volumes of high salt buffer, in the presence of detergent (1 M MgCI2, 0.01 M Tris HC1, pH7. 1!~,~ Triton XI00, 0.1 mg/ml baeitracin) (Bon and Massoulie, 19801. We prepared suspensions of particulate collagen-tailed forms in two different manners: (a) the tissues were homogenized in 10 volumes of low salt buffer (10raM Tris HCI, pH 7), (b} the supernatant of a high salt extraction (1 mM MgCI> 10mM Tris HC1, pH 7) was dialyzed against the low salt buffer. These suspensions were then diluted with 20 volumes of 10mM Tris HC1 pH 7 buffer, containing 0.1 mg/ml bacitracin, and various concen* l, 5- Bis(4- allyldimethylammoniumphenylt- pentane- 3 one dibromide (Sigma}.
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Fig. 1. Variations of acetylcholinesterase activity in the electric organs and electric lobes of Torpedo during embryonic development. The activity per gram of fresh tissue, determined by the method of Ellman et at. (196l) is plotted as a function of body length, electric lobes: electric organs: our data (@) and the values given by Krenz et al. (1980) (O) have been normalized for 80ram. The variations at early stages are shown with a larger scale in the inset.
Acetylcholinesterase forms in Torpedo embryos as the 35 mm body length stage. As shown in Fig. 1, the level of the enzyme remains low and approximately equal in both tissues until the 50 mm stage, when it reaches about 1% of the value observed at birth in the electric organs. As already reported by Krenz et al. (1980), acetylcholinesterase then increases rapidly, particularly in the electric organs, which after the 60 mm stage contain a much greater activity than the electric lobes. Sedimentation patterns of acetylcholinesterase, corresponding to different stages of development are illustrated in Fig. 2: they show that the increase in the enzyme activity is accompanied by complex variations of its composition in terms of molecular forms. The profiles given in Fig. 2 demonstrate that besides the A12 (17.5 S), Aa (13.8 S) and hydrophobic G 2 (6 S) forms, the tissues contain two molecular forms, sedimenting at 3.6S and 10.8 S, which have not been identified in detergent extracts of adult electric organs. These forms exist in the nervous tissue of adult Torpedo and appear to represent hydrophobic monomers (G1) and tetramers (G4) (Bon and Toutant, in preparation). The smaller A4 form is never abundant, but is detectable at the 50-60ram stages; it is clearly recognized in an aggregating fraction prepared from an extract of 58 mm electric organ (Fig. 3). From the sedimentation patterns of acetylcholinesterase, it is possible to estimate the contribution of each molecular form to the total activity. Figure 4 summarizes the variations of these proportions in the electric organs. The experimental points, representing the values obtained with different embryos, show a scatter of about 10%, because of experimental errors due to the incomplete resolution of the sedimentation patterns and probably also because of individual differences in the development of the embryos. The relative variations of the different forms are however quite clear. In the early stages of electric organ development, the hydrophobic G 2 and G4 forms are predominant, but A12 is already present; A12 then increases in proportion and is accompanied by As which transiently represents the most abundant form during the 50-60mm stages. G2 increases later: G2 and A12 are essentially the only forms present at the time of birth, G 2 being more abundant than A12. In the adult, A8 becomes visible again. By combining the relative proportions given in Fig. 4 with the evolution of the total activity in the electric organ, it is possible to evaluate approximately the absolute activity of each form during development. We have multiplied this value by the weight of the electric organ, obtained from Mellinger et al. (1978), and the results are given in Fig. 5, as a function of
579
body length. Considering that the growth of the embryos is approximately constant (1 mm/day) between the 30 and l l 0 m m stages (Krenz et al., 1980), these curves provide a rough approximation of the rate of accumulation of the different forms in the electric organs. They show that the activity increases markedly after the 60°70 mm stage, corresponding to an accumulation of A12, later followed and overtaken by an accumulation of G2. We did not construct a similar set of curves in the case of the electric lobes, which we have not investigated as extensively. As in the electric organs, Ga occurs together with G 2 and A12 at early stages but later decreases continually; A12 rapidly increases, becoming predominant around the 50mm stage. Its proportion transiently decreases, around the 800100 mm stages, as the smaller globular forms G1 and G 2 become relatively abundant (about 20°/0 of the total activity). The adult lobes contain a major proportion of A12, together with a small proportion of As. It is remarkable that As is not observed in the embryonic stages, but appears in the adult. (B) Solubilization and aooreoation properties of collagen.tailed forms in embryonic and adult Torpedo The analyses described in the previous section were performed with tissue extracts prepared in a high salt, detergent-containing medium, in order to solubilize quantitatively all forms of acetylcholinesterase. We also examined the influence of salt concentration on the solubility of the collagen-tailed forms in electric organs and electric lobes, hoping that it might reveal possible differences in their attachment to basal lamina or other structures. When the tissues were homogenized in 10mM Tris-HC1 pH 7, in the absence of divalent cations, a fraction of collagen-tailed acetylcholinesterase was recovered in the supernatant. This fraction was notably higher after a low speed centrifugation (Sorvall SS 34, 13,000rev/min, corresponding to 15,0009, 15min) than after a high speed centrifugation (Airfuge, maximal speed - 95,000 rev/min, corresponding to ~100,000g, 5min): we obtained respectively about 50 and 20% of the total activity in the case of the electric lobe. We found by sedimentation analysis in low salt sucrose gradients that in such electric lobes supernatants the collagen-tailed enzyme does not occur as individual molecules, but as potydisperse aggregates (around 50-70 S) explaining the variability of the yield of this low salt 'extraction'. In any case, it is clear that the fraction of these forms which is thus extracted in low salt is much smaller in the case of electric organs (5-10%) than in electric lobes.
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Fig. 3. Presence of the smallest asymmetric form (A+) in aggregates from a 58 mm embryo electric organ extract. A high salt extract of 58 mm embryo electric organ was fractionated into soluble and aggregated components after dialysis against a low salt buffer containing 40 mM MgCI2. The precipitated material was redissolved in high salt buffer prior to centrifugation in sucrose gradient (cfi Fig. 2). O O the soluble components correspond to the G+ and G 2 globular forms, g --Q the aggregated components correspond to the three asymmetric forms A12. As and clearly A,. The collagen-tailed molecules precipitate in the presence of a low concentration of divalent cations, and become solubilized by high concentrations of MgCIz (Fig. 6A). We compared the influence of MgCi2 concentration in two different experiment~il conditions: the solubilization of acetylcholinesterase from the particulate fraction of a tissue homogenate which had been prepared in low salt, and the dissociation of aggregates which had been precipitated by dialysis of a high salt solubilized extract against a low salt buffer. In the case of the electric lobes, we obtained essentially the same curve for the low speed 'soluble' and 'insoluble' fractions resulting from homogenization of the tissue in 10 mM Tris-HCl buffer, as well as for high salt solubilized, reaggregated enzyme. The dissociation of reaggregated collagen-
tailed forms from electric organ was achieved at a much smaller MgC12 concentration, than that required from their dissociation from the tissue homogenate indicating that interactions in the native structures differ from those which occur in aggregates obtained in vitro. The solubilization curves of the electric organ enzyme (but not the dissociation of reaggregated enzyme) in fact depends upon the developmental stage: Fig. 6(B) shows that the collagen-tailed enzyme becomes more difficult to solubilize as the electrocytes mature. It must finally be noted that the collagentailed forms possess identical sedimentation coefficients, and show their characteristic sensitivity to collagenase at all developmental stages, suggesting that they have the same molecular structure.
Fig. 2 (opposite). Sedimentation patterns of acetylcholinesterase at various developmental stages. (A)-(D): electric organs; (E)-(H): electric lobes. Tissue extracts were centrifuged in sucrose gradients (see "Experimental Proc6dures"). The acetylcholinesterase activity is plotted on an arbitrary scale, as a function of the fraction number. Migration is from the right to the left. The curves have been normalized to the same maximal value, and the corresponding forms brought into coincidence. The positions of the sedimentation standards //-galactosidase (16 S), catalase (11.3 S) and alkaline phosphatase (6.1 S) are indicated by arrows in frames A and E. The embryo length is indicated in the upper right corner of each graph.
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DISCUSSION In this study, we analyzed the molecular forms of acetylcholinesterase from Torpedo embryonic electric organs and electric lobes homogenized in a medium containing 1 M MgClz and 1°41 Triton XI00, which allows the complete solubilization of b o t h collagen-
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Fig. 5. Evolution of the total activity of acetylcholincsterase molecular forms in Torpedo electric organs, during embryonic development. "'- G2:--- G 4 : A~ and A~z. The activity per gram of electric organ was evaluated by applying the proportions determined in Fig. 2 to the total activity (Fig. 1). The result was multiplied by the weight of the electric organ, obtained from the curves given by Mellinger et al. (1978). The evolutions of the minor G 4 and A~ forms arc shown with a larger scale in the inset.
tailed and globular forms of the enzyme, including its hydrophobic fraction. We thus observed the existence of m o n o m e r i c and tetrameric hydrophobic acetylcholinesterase which had not yet been identified as native forms in detergent extracts of adult electric organs. We recently demonstrated that in addition to the 6 S dimeric form, nervous tissues from adult Torpedo contain collagenase-insensitive hydrophobic forms of acetylcholinesterase, which correspond to monomeric (3.6S) and tetrameric (9.8S) molecules (Bon and Toutant, in preparation). Torpedo tissues therefore contain three globular forms, homologous to the G~, G 2 and G+ forms observed in higher vertebrates (Bon et al., 1979; Grassi, Vigny and Massouli6, 1982).
Acetylcholinesterase forms in Torpedo embryos One aim of this study was to examine whether the different forms appear in a definite sequence, which might correspond to progressive stages in their biosynthesis. If this were the case, G1 and G2 would have been expected to precede G4. On the contrary, G4 is found as a major component of acetylcholinesterase at early stages (before the 50 mm stage) but then decreases steadily, in both the electric organs and the electric lobes. In the electric organs, G~ has not been identified at any stage, and hydrophobic G 2 passes through a minimum, in relative terms, in 60-70 mm stage embryos. In the electric lobes, the proportion of G1 passes through a maximum, around the 80-100mm stage. Clearly these variations cannot be simply related to biosynthetic relationships between the three globular forms. In a similar manner, it is not possible to consider the hydrophobic G2 form, which represents a high proportion of enzyme in the adult electric organs, as a pool of precursors of the collagen-tailed forms, since A~2 accumulates earlier than G2. It is not possible at the moment to decide whether distinct genes direct the synthesis of globular and collagen-tailed acetylcholinesterase, or whether the assembly lines leading to the two types of molecules are organized in such a way that biosynthetic intermediates do not normally accumulate in a conspicuous manner. As for the collagen-tailed forms, the most salient feature is probably the transient appearance of A4 and A8 forms in the electric organs about the 55 mm stage. As indicated in Fig. 4, the highest specific activity of the Aa form corresponds to the onset of the rapid increase of A12 (60-100mm). This of course suggests that As might represent an intermediate in the assembly of At2, and that its relatively high level would be directly correlated with an active synthesis of the larger collagen-tailed form. The fact that As was not observed in the electric lobes during the embryonic stages may be related to the lower rate of A~2 synthesis in the nervous tissue. It is also remarkable that although As is absent at birth, it occurs at the adult stage both in the electric organs and in the electric lobes. It may be that the A forms represent different molecular structures at different stages since it has recently been shown that catalytic subunits may be partly replaced to variable degrees by non-catalytic subunits of unknown function (Lee et al., 1982). We must now examine whether any correlations can be established between the differentiation of the electrocytes, particularly their innervation by electric lobe motoneurons, and the appearance of specific forms of acetylcholinesterase. The studies of Mellinger et al. (1978) and Fox and Richardson (1978, 1979)
583
have established that the development of the electric organ can be divided in two phases: a myogenic phase and an electrogenic phase. During the myogenic phase, i.e. until the 40 mm stage, the cells possess the characteristics of embryonic myotubes, they contain myofibrils, and are vertically oriented. Between the 40 and 55 mm stages, the cells become horizontally flattened and lose their organized myofibrils. After the 55 mm stage, the level of acetylcholine receptor increases strongly (Krenz et al., 1980). Nerve terminals may be seen in the electric organs after the 60-70 mm stages, but only extremely small electrical responses can be elicited at these early stages by stimulating the electric nerve. Electric organ discharge amplitude begins to increase exponentially after the 90 mm stage and reaches adult values at birth. Studies of acetylcholinesterase forms during the development of rat and chicken have established that globular forms exist very early in skeletal muscles but that the appearance of collagen-tailed forms is concomittant with the establishment of neuromuscular contacts (Massouli6 et al., 1980; Massouli6 and Bon, 1982). It was therefore surprising to find that the A12 form already represents a significant proportion of acetylcholinesterase in the electric organs at the earliest stage which we investigated (35 mm) i.e. before the end of the myogenic phase of development and before synapse formation begins. The collagen-tailed forms of acetylcholinesterase therefore do not appear to be controlled in the same manner in the Torpedo electric organs as in the muscles of higher vertebrates. However, it is interesting to note that the A~2 form starts to increase rapidly in absolute amount at the 55-60mm stage, i.e. when the electrocytes become morphologically differentiated and when the ingrowth of electromotor nerves into the inter electrocyte space begins. It is probably relevant that the basal lamina following the ventral face of the electrocytes is already visible at this stage (Richardson, 1980), since the collagen-tailed forms are thought to be localized in these extracellular structures. It is remarkable that the major accumulation of G2, which occurs later than that of A~2 coincides with the major development of nerve terminals and with the period of appearance and increase of electric organ discharge amplitude (Krenz et al., 1980), in agreement with recent findings which indicate that this form is largely presynaptic (Morel and Dreyfus, 1982; Li and Bon, 1982). This suggests an important physiological significance of G2 acetylcholinesterase at the presynaptic sites. It may be surprising, in this view, that G2 never reaches very high levels in the electric lobes, where A12 in fact becomes largely predominant
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in the adult, The cytochemical distribution of acetylcholinesterase activity in the cell body of the motor neurons (Tsuji, 1977) suggests that this A~ 2 enzyme is mostly intracellular, in contrast with the electric organs. The characteristics of solubilization of the collagentailed forms are indeed different in the electric organs and electric lobes, in two respects. Firstly, the proportion of collagen-tailed acetylcholinesterase which is recovered in the low speed supernatant after a low salt homogenization is markedly higher in the electric lobes, as also observed by W i t z e m a n n and Boustead (1981). We have shown that this enzyme, in fact. represents polydisperse aggregates rather than isolated molecules, so that the yield of low salt 'extracted' collagen-tailed acetylcholinesterase markedly depends upon the centrifugation conditions. Secondly, the solubilization of collagen-tailed forms of acetylcholinesterase by salt requires a higher concentration of MgCI2 in the electric organs than in the electric lobes where it followed the curve observed for dissociation of previously solubilized, in ritro reaggregated enzyme. Furthermore, the solubilization of collagentailed enzyme from electric organs occurs at increasingly high MgCI: concentrations, during the course of maturation. These variations do not appear correlated with intrinsic differences in the collagen-tailed molecules themselves, and in particular their aggregation properties appear similar, as indicated by the dissociation of in t:itro aggregated enzyme. The distinct solubilization properties of these enzymes in electric lobes and electric organs therefore probably result from their intracellular localization in the first case, and the evolution of more complex and stable associations within the developing basal lamina in the second case. In conclusion, the present study revealed that during the development of the electric organs and the electric lobes of Torpedo, the different molecular forms of acetylcholinesterase present a complex evolution which is not readily correlated with biosynthetic sequences, but rather reflects the morphological and physiological differentiation of the nervous and electric tissues.
Acknowledgements I wish to thank particularly Dr. Guy Richardson, who provided the Torpedo embryos, for his help with the dissections, and for very useful advice. I am grateful also to Dr. Jean Massouli6 for his encouragement throughout the course of the work and for helpful discussions, and to Pierre Allemand for his invaluable technical assistance. This work was supported by grants of the Centre National de la Recherche Scientifique, and the Direction Gdn6rale h la Recherche Scientifique et Technique.
REFEREN('):S Bon, S., Huet, M., Lemonnier, M., Rieger, I~. and Massouli& J, (1976L Molecular forms of l£1ectrophoru~ acclylcholinesterase. Eur, J. Biochem. 6g, 523 53/), Boil, S., Cartaud, J. and Massoulie, J. (1978!. The dependence of acetylcholinesterase aggregation at low ionic strength upon a polyanionic component. Era'. I. Biochem. 85. 1 14. Bon, S., Vigny, M. and Massoulie. J. (1979). Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. hath. Acad. Sci. U.S.A. 76, 2546 2550. Bon, S. and Massoulie. J. (19801, Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organs. Proc. hath. :lead. Sei. ('.S.A. 77, 4464 4468. Etlmann. G. L., Courtney, K. D., Andres, V. and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmac. 7, 88-95. Fox. C. Q. and Richardson, G. P. (1978). The developmental morphology of Torpedo marmorata: electric organmyogenic phase, J. comp. Neurol. 179, 677 697. Fox. G. Q. and Richardson. G. P, (19791. The developmental morphology of Torpedo marmorata: electric organelectrogenic phase. J. eomp. Nem'ol. 185, 293 314. Grassi. J., Vigny, M. and Massouli6, J. (1982), Molecular forms of acetylcholinesterase in bovine caudate nucleus and superior cervical ganglion: solubility properties and hydrophobic character. J. Neuroehem. 38, 457 469 Krcnz, W. D.. Tashiro, T,, WS.chtler. K., Whittaker. V. P. and Witzemann. V. (1980). Aspects of the chemical embryology of the etectromotor system of Torpedo marmorata with special reference to synaptogenesis, ~'euroscience 5, 617 624. Lec. S.. Camp, S. and Taylor, P. (1982i. Structural characterization of the asymmetric (17S + 13 S) species of acetylcholinesterase from Torpedo: analysis of subunit composition. 3. biol. Chem. in press. Li, Z. Y. and Bon, C. (1982). Presence of a membranebound acetylcholinesterase form in a nerve-endings preparation from Torpedo marmorata electric organ, J. Neurochem. in press. Lwcbuga-Mukasa, J. S.. Lappi, S. and Taylor, P. 11976). Molecular forms of acetylcholinesterase from Torpedo calilorniea: their relationship to synaptic membranes. Biochemi,~try 15, 1425 1234. Massouli6, J., Bon, S. and Vigny, M. (1980). The polymorphism of cholinesterases in vertebrates. Neuroehem. Int. 2. 161 184. Massouli,L J. and Bon, S, (1982). The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. Ann. Rer, Neurosci. 5, 57 106. Mellinger, J., Belbenoit. P., Ravaillc, M. and Szabo. 1-. (I978). Electric organ development in Torpedo marmorata, Chondrichthyes. Dec. Biol. 67, 167 188. Morel, N. and Dreyfus, P. (1982). Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ. Neurochemistry Int. 4, 283 288. Richardson. G. P. (19801. Development of the eleclromotor system of Torpedo nlarnun'ata. PhD, Thesis, University of Sussex. Tsuji. S. (19771. Acetylcholinesterasc positive elcctromotor
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