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The molecular weight and subunit structure of acetylcholinesterase preparations from the electric organ of the electric EEL
Vol. 59, No. 1, 1974
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE M O L E C U L A R WEIGHT AND SUBUNIT S T R U C T U R E OF ACETYLCHOLINESTERASE PREPARATIONS FROM THE ELECTRIC O R G A N OF T H E ELECTRIC E E L Yadin Dudai and Israel Silman
Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel Received May 13,1974 SUMMARY: Acetylcholinesterase (ACHE) preparations obtained from electric organ tissue of Electrophorus elec~ricus subsequent to tryptic digestion and/or autolysis had sedimentation coefficients of about 11 S and molecular weights of 320,000-350,000. These values did not significantly decrease upon prolonged autolysis. The major polypeptide in 11 S AChE had molecular weights of B2,000 "~ 6,000 and 59,000 • 4,000. The ratios of these two components was a function of the degree of autolysis of the tissue from which the enzyme was purified. In less au~olysed tissue the principle component was the 82,000 one, while after prolonged autolysis the 59,000 component predominated. The appearance of the 59,000 component was accompanied by the appearance of polypeptides of ~ 25,000. It is proposed that autolysis and/or proteol y s i s cleave the 82,000 polypeptide chain of native ACHE into fragments of 59,000 and -,, 25,000 which are retained in the quaternary structure of the enzyme unless it is denatured. Possible models for the quaternary structure and arrangement of disulfide bridges in 11 S AChE are discussed. Acetylcholinesterase (ACHE) can be obtained from electric organ tissue of the electric eel, either by prolonged autolysis or by controlled tryptic digestion, in a molecular form with a sedimentation coefficient of about 11 S (1,2). This f o r m is apparently a degradation product of the large asymmetric "native" forms of the enzyme present in fresh tissue (3,4).
The molecular weight of 11 S AChE preparation'~
obtained in several laboratories has been reported to be 230,000-260,000 (5-7),
and values of 64,000 (6) and 42,000 (7) were reported for the molecular weight of the subunits of such preparations. We earlier reported a molecular weight o f ~ 335,000 (4) for preparations of 11 S AChE purified from fresh or partially autolysed electric organ tissue by affinity chromatography (2), and also presented evidence that the major polypeptide component, which contains the active site of the enzyme, has a molecular weight of about 80,000 (2, 8,9).
We also observed a minor active-site-containing polypeptide
of molecular weight about 60,000 as well as traces of polypeptide chains of much lower molecular weight.
In the present study the molecular properties of an 11 S
AChE preparation purified from extensively autolysed tissue are examined, and it is shown that the relative amounts of the major polypeptide components of elecLric eel
2opyright © 1974 by Academic Press, Inc. 411 rights o/reproduction in any ]orm reserved.
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A C h E are dependent on the history of the tissue preparation, The 80,000 polypeptlde is the major component in enzyme purified from fresh tissue, while the lower molecular weight components are present in increasing amounts in tissue which has undergone prolonged autolysis. However, conversion of the 80,000 subunit to smaller polypeptide components is not accompanied by a significant reduction in the molecular weight of the enzyme.
On the basis of these data the quaternary structure of ii S A C h E
and the discrepancies between the results obtained by different groups will be discussed.
EXPERIMENTAL AChE Preparations:
Purified 11 S AChE was obtained either following tryptic
digestion of fresh tissue, partially autolysed tissue and extensively autolysed tissue, or from partially autolysed tissue without tryptic digestion. The tissue was autolysed by storage under toluene at 40 for prolonged periods as described by Rothenberg and Nachmansohn (13). The various preparations were purified by affinity chromatography as described previously (2). The preparations obtained by tryptic digestion of fresh tissue and partially autolysed tissue (i. e. tissue which had been stored under toluene at 4° for periods ranging from several weeks to 2 years) correspond to AChE A and AChE B respectively in Ref. 2.
The preparation obtained by tryptic digestion of
extensively autolysed tissue (which had been stored under toluene at 40 for about 4 years) was purified by the same procedure employed for purification of AChE B, and will be called AChE B'.
The preparation obtained from partially autolysed tissue
without tryptic digestion corresponds to AChE C in Ref. 2,
"Native" forms of AChE
(i. e. 18 S + 14 S ACHE) were also purified by affinity chromatography as described
previously (9). Labelling of A C h E
with 3H-diisopropylnuorophosphate (3H-DFP; 0.9 Ci/mmo[
in propylene glycol, N e w England Nuclear, Boston) and treatment with pyridine 2aldoxime methiodide (2-PAM) were performed as described earlier (8). Enzyme assays and sucrose gradient centrifugation were performed as previously described (2). Ecluilibriu.m " sedimentation experiments, by the high speed meniscus depletion method of Yphantis, were performed as before (4). Polyacrylamide gel eleetrophoresis in the presence of SDS,with and without ~-mercaptoethanol, was performed as described previously (2,8). 12 c m gels were used for molecular weight calibration. 3H-DFP-labelled A C h E was electrophoresed similarly and the labelled peaks monitored as before (8).
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RESULTS AND DISCUSSION Three different preparations of AChE (A, B' and C), purified from electric organ tissue after different degrees of tryptic digestion and/or autolysis, were subjected to equilibrium sedimentation. Fig. 1.
The molecular weights obtained are shown in
All three preparations appear to be quite homogeneous, as can be deduced
I
3.8
I
I
I
I
I
I
I
I
I
I--
J
I
[
]
F---
I 600
I
I 800
(I)
3~ 34
32 I
I
~
(2) '0 38 x
~ 35 ~9
~ a4 ~32 o
I
I
[ ~ I F - - -
(3) ~ 38 3.6 54
.i ?"~?,?t-"
32 I 200
[
~
I 400
I
FRINGE DISPLACE M ENT,/z
Figure 1. The weight-average molecular weigbi of v a r i o u s 11 S AChE preparations, plotted against fringe displacement as computed by a computer high-speed equilibrium ultracentrifugation program (4). (1) Acetyleholinesterase A; (2) ace~yloholinesterase C; (3) acetylcholtnesterase B'. All ~ r e e preparations were centrifuged at 10,000 rpm, as described i n t h e text.
from the small variation of the molecular weight plotted vs. fringe displacement (whieh is proportional to concentration) along the ultracentrifuge cell. The preparations have molecular weights in the range of 320,000-350,000, assuming a v of 0. 723
(4).
These values are all significantly higher than the molecular weight of
230,000-260,000
previously reported for AChE preparations with a sedimentation
coefficient of about 11 S (5-7). AChE-B', prepared from extensively autolysed tissue, displays the same specific activity and sedimentation coefficient as AChE-A, AChE-B and AChE-C.
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The molecular weights of the potypeptide components of all four 11 S preparations were determined by performing polyacrylamide gel electrophoresis, in the presence of
SDS and ~-mercaptoethanol.
They all contain two main polypeptide components with
molecular weights of 82,000 + 6,000 and 59,000 ± 4,000, respectively.
However,
whereas in the "native" forms of AChE (18 S + 14 S, Ref. 9) the 82,000 polypepLide
is the major component and the 59,000 polypeptide is present only in small amounts, if at all
(Fig.
2.1,
Ref. 9), ~he relative amounts of the two polypeptides varies
markedly in different 11 S acetylcholinesterase preparations (Fig. 2.2
82 59,000-
and 2.3).
In
Q
25,000-
CI)
C2)
C3)
Fib,ure 2. Acrylamide gel electrophoresis, in the p r e s e n c e of 8DS and ~-mercaptoethanol, of AChE preparations. 50/0 acrylamide gels were used, and stained with Coomassie blue. (1) "Native ' AChE (14 S + 18 SI; (2) AChE~B; (3) AChE-B'.
11 S AChE derived from tissue which had undergone the most extensive autolysis, (AChE-B'), the relative amount of the 82,000 component is reduced and the amount of the 59,000 component is markedly increased (Fig. 2.3).
The increase in the relative
amount of the 59,000 component is accompanied by the appearance of polypeptides of lower molecular weight (~25,000).
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Both the 82,000 and 59,000 components o b s e r v e d b y staining for p r o t e i n contain D F P - b i n d i n g s i t e s (Fig. 3).
Again it can be seen that the r e l a t i v e amount of the
59,000 component, a s r e v e a l e d by D Fl:Lbinding, i n c r e a s e s in the e x t e n s i v e l y a u t o l y s e d 11 S A C h E - B ' (Fig. 3.3).
We found that about 90°/0 of the r a d i o a c t i v e d i i s o p r o p y l -
/ 5
/
,
(2)
• ~59,ooo
~s2,oo%
~0 6 × 4 o_
,000
o~59,000
(3) 50 20~
~82,oooo
0
I0
~ i
20
! 30
40
50
MIGRAT]ON (millimeter)
F i g u r e 3. A c r y l a m i d e gel e l e c t r o p h o r e s i s , in the p r e s e n c e of SDS and ~ - m e r c a p t o ethanol, of 3 H - D F P - l a b e l l e d AChE s a m p l e s . The g e l s were sllced~ r a d i o a c t i v i t y m e a s u r e d and the m o l e c u l a r weigl~s c a l i b r a t e d a s d e s c r i b e d u n d e r Methods. (1) "Native" AChE (14 S + 18 S); (2) AChE B; (3) AChE B ~.
p h o s p h o r y l g r o u p s could be r e m o v e d f r o m boLh components with the s p e c i f i c a c e t y l c h o l i n e s t e r a s e r e a c t i v a t o r 2 - P A M (14) under the conditions d e s c r i b e d by us (8}.
No
r a d i o a c t i v i t y a p p e a r e d to be a s s o c i a t e d w~h the f a s t e r - m o * 4 n g (,~25,000) p o l y p e p t i d e components, indicating that these p o i y p e p t i d e s do not contain active s i t e s . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s in the p r e s e n c e of SDS but in the a b s e n c e of ~ - m e r c a p t o e t h a n o l r e v e a l s that a l l the p r e p a r a t i o n s of 11 S AChE d i s p l a y polypeptides o f 1 6 8 , 0 0 0 4- 8,000 and 88,000 4- 4~000, both of which bind D F P .
Itthus seems
p l a u s i b l e t o a s s u m e that the 160; 000 - !70~ 000 component is a d i m e r i c s t r u c t u r e in which two subunits a r e connected by i n t e r s u b u n ~ disulfide bonds (8).
This p o s s i b i l i t y
was a l s o p r o p o s e d b y F r o e d e and Wilson (15) on the b a s i s of s u c r o s e - g r a d i e n t c e n t r i -
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fugation e x p e r i m e n t s employing 14C-DFP-labet~ed AChE tx,eated e i t h e r with guanidine alone o r with guanidine and ~ - m e r c a p t o e t h a n o L The s e d i m e n t a t i o n eq~i~ibrium and a c r y ! a m i d e gel e l e c t r o p h o r e s i s data~as well as the r e s u l t s of the d e t e r m i n a t i o n s of s values and s p e c i f i c a c t i v i t i e s of the v a r i o u s 11 S AChE p r e p a r a t i o n s ~ can be b e s t explained by a s s u m i n g that a u t o l y s i s can lead to cleavage of the 82~000 peptide chains p r e s e n t in the native enzyme m o l e c u l e , at a l i m i t e d n u m b e r of s u s c e p t i b l e s i t e s .
However, this cleavage does not l e a d to r e l e a s e
of the 59,000 and-.~ 25,000 m o l e c u ) a r weight f r a g m e n t s p r o d u c e d and to d i s r u p t i o n of the q u a t e r n a r y s t r u c t u r e , ur~_~ess the enzyme is denatured.
S e v e r a l g r o u p s have
r e p o r t e d c a s e s in which p r o t e o l y t i c clean,-age p e r s e does not change the o v e r a l l m a s s and q u a t e r n a r y s t r u c t u r e of p r o t e i n s (16~19} and we have a l r e a d y s u g g e s t e d this p o s s i b i l i t y for AChE (2). On the b a s i s of the above, va~fous m o d e l s can be s u g g e s t e d for the q u a t e r n a r y s t r u c t u r e of 11 S AChE. Since ~:native ~r (Le. 18 S + 14 S} p r e p a r a t i o n s of AChE and s o m e 11 S p r e p a r a t i o n s contain r e i a t i v e l y sma}Z amounts of the 80,000 component on SDS e l e c t r o p h o r e s i s in the a b s e n c e of ~-mercaptoethanol0 it s e e m s unlikely that a valid m o d e l will include subunits unl-iffked to any o t h e r sub~mit by disulfide b r i d g e s . We will c o n s i d e r , t h e r e f o r e , on~_y m o d e l s in which p a i r s of subunits a r e linked b y d i s u l f i d e bonds. Two s i m p i e m o d e l s az'e shoa, n in Fig. 4.
In Model_ I the subunits
a r e linked by disulfide bridges~ as aye the two pa~ts of the polypeptide chain which a r e cleaved b y p r o t e o l y s i s o r a u t o I y s i s . The pol-ypeptides a r e a r r a n g e d in p a r a l l e l (i. e. with the s a m e p o l a r i t y f r o m the NH2~terminal to the COOH-terminal residue} as is the c a s e in y - g l o b u l i n (20}.
This m o d e i does not s e e m v e r y s a t i s f a c t o r y , b e c a u s e
w h a t e v e r the p e r c e n t a g e of subunits cleaved e n z y m i c a l l y , one would not e x p e c t a p p e a r a n c e of polypeptides of m o l e c u l a r weight 80,000 on a e r y l a m i d e g e l s i n the a b s e n c e of ~ - m e r c a p t o e t h a n o l , and this is n o t in fact the c a s e (8).
Another s i m p l e
model, Model 12, does not r e q u i r e i n t r a s u b u n i t d i s u l f i d e s and p o s t u l a t e s an a n t i p a r a l l e l a r r a n g e m e n t of polypeptide chains linked by disulfide b r i d g e s in each of which one c y s t e i n e r e s i d u e is beyond the cleavage point.
A n t i p a r a l l e l a r r a n g e m e n t of
polypeptide chains in multisubunit p r o t e i n s h a s indeed been d e s c r i b e d in s e v e r a l e a s e s (21).
This m o d e l is m o r e a t t r a c t i v e s i n c e it can account for the a p p e a r a n c e of
a-,~80,000 s p e c i e s even p r i o r to r e d u c t i o n .
O b v i o u s l y , m o r e complex m o d e l s can
account for the d a t a a s s u m i n g m o r e disulfide b r i d g e s a n d / o r m o r e c l e a v a g e p o i n t s . M o r e o v e r , 11 S AChE is i t s e l f a d e g r a d a t i o n product of "native"'AChE (1,2}, and the q u a t e r n a r y s t r u c t u r e of the "native", f o r m s of the enzyme m u s t take into account i n t e r -
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
COOH
COOH
A
k
COOH
A
]I
COOH
NH2
,k
COOH
'1
NH 2
-S--S-
-S--S-
i
-S--S- 1
t NH 2
¢
NH 2
NH2
y
i1'
3~'0-350,000
MW:
COOH
'~I
!
4TM
:y - S - - S - ¢,I
-S--S- i
MW:
NH2
COOH
NH 2
320-350,000
~Hz
C00H
A,
-S--S-
J I
COOH
NH 2
COOH
,k
COOH
.H~ C~OH
i -s-s-Ck
,b_ -s
-S--S-
¢1 NH 2
MW:
-S-S-
1"
Y COOH
NH2
~165~000 COOH
.s
80,000
COOH
.I,
NH2
MW: COOH
C00H
SH
qM'
HS-
'~#v
HS -
HS
~ SH
I}
- S H HS -
4 ..
,I' NH2
MW:
HS-
l' NH2
¢ NH2
~ eo,ooo; ~6o,ooo;~ 25000
MW:
I~
-SH
NH 2
~ 8o,ooo;~6o,0oo,~ 25,000
Figure 4. Schematic models for the subunit structure of 11 S AChE~ together with the products to be expected under the conditions employed for SDS-acrylamide gel electrophoresis, both in the presence t~nd absence of [~-mercaptoethanol, subsequent to cleavage of either one or both polypeptides of the dimer. The site where a cleavage can occur i s indicated by a serrated line. For discussion s e e text.
actions between larger numbers of subunits, and also Lhe presumably specific mode of attachment of the "tail" of the native enzyme to the multisubunit head (3,4).
However,
any model should take into account the fact that reducing agents are n e c e s s a r y for complete dissociation of the AChE molecule into its subunits, and for r e l e a s e of fragments from the cleaved subunits, subsequent to proteolysis or autolysis. Thus our results indicate that 11 S AChE is a tetramer of molecular weight 320,000-350,000, each subunit being of about 80,000.
Our observation that the
principal subtmits present in electric eel AChE have molecular weights of about 80,000 and 60,000 is in agreement with recent results from other laboratories (10-12). Our r e s u l t s also indicate that p01ypeptides of about 60,000 and 25,000 appear gradually following autolysis or tryptic digestion, but that these polypeptide fragments, formed by cleavage of the 80,000 subunit, are not released from the molecule unless it is denatured.
Similar conclusions have r e c e n t l y been reached by Chen et al. (22) on the
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
basis of acrylamide gel electrophoresis and exclusion chromatography in 6 M guanidine solutions. Leuzinger et al. (6) reported earlier that 11 S AChE is a t e t r a m e r of 260,000, containing four 64,000 subunits.
We cannot exclude the possibility that under certain
conditions cleavage of the 80,000 polypeptide is followed by r e l e a s e of polypeptide fragments, even p r i o r to denaturation. Such a process could result in formation of an active AChE t e t r a m e r of 230,000-260,000, as reported previously (5-7), with subunits of 64,000 or less (6,7).
However, we did not observe the formation of such a species
in our preparations.
Acknowledgments The support of the Volkswagen Foundation is gratefully acknowledged. We wish to thank Mrs. Esther Saksel-Roth for her skilful technical assistance and Drs. Martin Hirshfeld and Robert Josephs for help with the ultracentrifuge experiments. REFERENCES
1. 2. 3.
Massouli~, J . , and Rieger, F. (1969} Eur. J. Biochem. 1__~1,441-455. Dudai, Y., Silman, I., Kalderon, N., and Blumberg, S. (1972) Biochim. Biophys. Acta, 268, 138-157. ./ Rieger, F . , Bon, S., Massouhe, J., and Cartaud, J. (1973) Eur. J. Biochem.
3~, 539-547. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Dudai,Y., Herzberg, M., and Silman, I. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2473-2476. Kremzner, L.T., andWilson, I.B. (1964) Biochemistry, 3, 1902-1905. Leuzinger, W., Goldberg, M., and Cauvin, E. (1969) J. Mol. Biol. 40, 217-225. Millar, D.B., and Grafius, M.A. (1970) FEBS Lett. 1._22, 61-64. Dudai,Y., and Silman, I. (1971) FEBS Lett° 1_6_6,324-328. Dudai,Y., Silman, I., Shinitzky, M., and Blumberg~ S. (1972) Proc. Natl. Aead. Sci. U.S.A. 6__99,2400-2403. Berman, J.D. (1973) Biochemistry, 1_22, 1710-1715; Bock, E., Chen, Y. T . , and Rosenberry, T . L . (1973) Fed. Proc. 322, 510. Powell, J . T . , Bon, S,, Rieger, F . , and Massoul~d~ J. (1973) FEBS Lett. 36, 17-21. Rothenberg, M . A . , and Nachmansohn, D. (1947) J. Biol. Chem. 168, 223-231. Wilson, I . B . , Ginsburg, S . , and Quan, C. (1958) Arch. Biochem. Biophys. 7._7_7, 286-296. Froede, H . C . , and Wilson, I.B. (1970) Israel J. Med. Sci. 6, 179-184. Ullmann, A . , Jacob, F . , and Monod, J. (1968) J° MoL Biol. 3.~2~ 1-13. Goldberg, M.E. (1969) J. Mol. Biol. 4._66, 441-446. Labeyrie, F . , andBaudras, A. (1972) Eur. J. Biochem. 2_55, 33-40. Jacq, C., and Lederer, F. (1972) Eur. J. Biochem° 2.~5~41-48. Gall, W . E . , Cunningham, B . A . , Waxdal, M . J . , Konigsberg, W . H . , and Edelman, G.M. (1968) Biochemistry, 7, 1973-1982. Matthews, B.W., and Bernard, S,A. (1973) Ann. Rev. Biophys. Bioeng. 2, 257-312. Chen, Y. T., Rosenberry, T . L . , and Bock, E. (1974} manuscript in preparation.
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THE M O L E C U L A R WEIGHT AND SUBUNIT S T R U C T U R E OF ACETYLCHOLINESTERASE PREPARATIONS FROM THE ELECTRIC O R G A N OF T H E ELECTRIC E E L Yadin Dudai and Israel Silman
Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel Received May 13,1974 SUMMARY: Acetylcholinesterase (ACHE) preparations obtained from electric organ tissue of Electrophorus elec~ricus subsequent to tryptic digestion and/or autolysis had sedimentation coefficients of about 11 S and molecular weights of 320,000-350,000. These values did not significantly decrease upon prolonged autolysis. The major polypeptide in 11 S AChE had molecular weights of B2,000 "~ 6,000 and 59,000 • 4,000. The ratios of these two components was a function of the degree of autolysis of the tissue from which the enzyme was purified. In less au~olysed tissue the principle component was the 82,000 one, while after prolonged autolysis the 59,000 component predominated. The appearance of the 59,000 component was accompanied by the appearance of polypeptides of ~ 25,000. It is proposed that autolysis and/or proteol y s i s cleave the 82,000 polypeptide chain of native ACHE into fragments of 59,000 and -,, 25,000 which are retained in the quaternary structure of the enzyme unless it is denatured. Possible models for the quaternary structure and arrangement of disulfide bridges in 11 S AChE are discussed. Acetylcholinesterase (ACHE) can be obtained from electric organ tissue of the electric eel, either by prolonged autolysis or by controlled tryptic digestion, in a molecular form with a sedimentation coefficient of about 11 S (1,2). This f o r m is apparently a degradation product of the large asymmetric "native" forms of the enzyme present in fresh tissue (3,4).
The molecular weight of 11 S AChE preparation'~
obtained in several laboratories has been reported to be 230,000-260,000 (5-7),
and values of 64,000 (6) and 42,000 (7) were reported for the molecular weight of the subunits of such preparations. We earlier reported a molecular weight o f ~ 335,000 (4) for preparations of 11 S AChE purified from fresh or partially autolysed electric organ tissue by affinity chromatography (2), and also presented evidence that the major polypeptide component, which contains the active site of the enzyme, has a molecular weight of about 80,000 (2, 8,9).
We also observed a minor active-site-containing polypeptide
of molecular weight about 60,000 as well as traces of polypeptide chains of much lower molecular weight.
In the present study the molecular properties of an 11 S
AChE preparation purified from extensively autolysed tissue are examined, and it is shown that the relative amounts of the major polypeptide components of elecLric eel
2opyright © 1974 by Academic Press, Inc. 411 rights o/reproduction in any ]orm reserved.
117
Vol. 59, No. 1, 1974
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A C h E are dependent on the history of the tissue preparation, The 80,000 polypeptlde is the major component in enzyme purified from fresh tissue, while the lower molecular weight components are present in increasing amounts in tissue which has undergone prolonged autolysis. However, conversion of the 80,000 subunit to smaller polypeptide components is not accompanied by a significant reduction in the molecular weight of the enzyme.
On the basis of these data the quaternary structure of ii S A C h E
and the discrepancies between the results obtained by different groups will be discussed.
EXPERIMENTAL AChE Preparations:
Purified 11 S AChE was obtained either following tryptic
digestion of fresh tissue, partially autolysed tissue and extensively autolysed tissue, or from partially autolysed tissue without tryptic digestion. The tissue was autolysed by storage under toluene at 40 for prolonged periods as described by Rothenberg and Nachmansohn (13). The various preparations were purified by affinity chromatography as described previously (2). The preparations obtained by tryptic digestion of fresh tissue and partially autolysed tissue (i. e. tissue which had been stored under toluene at 4° for periods ranging from several weeks to 2 years) correspond to AChE A and AChE B respectively in Ref. 2.
The preparation obtained by tryptic digestion of
extensively autolysed tissue (which had been stored under toluene at 40 for about 4 years) was purified by the same procedure employed for purification of AChE B, and will be called AChE B'.
The preparation obtained from partially autolysed tissue
without tryptic digestion corresponds to AChE C in Ref. 2,
"Native" forms of AChE
(i. e. 18 S + 14 S ACHE) were also purified by affinity chromatography as described
previously (9). Labelling of A C h E
with 3H-diisopropylnuorophosphate (3H-DFP; 0.9 Ci/mmo[
in propylene glycol, N e w England Nuclear, Boston) and treatment with pyridine 2aldoxime methiodide (2-PAM) were performed as described earlier (8). Enzyme assays and sucrose gradient centrifugation were performed as previously described (2). Ecluilibriu.m " sedimentation experiments, by the high speed meniscus depletion method of Yphantis, were performed as before (4). Polyacrylamide gel eleetrophoresis in the presence of SDS,with and without ~-mercaptoethanol, was performed as described previously (2,8). 12 c m gels were used for molecular weight calibration. 3H-DFP-labelled A C h E was electrophoresed similarly and the labelled peaks monitored as before (8).
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RESULTS AND DISCUSSION Three different preparations of AChE (A, B' and C), purified from electric organ tissue after different degrees of tryptic digestion and/or autolysis, were subjected to equilibrium sedimentation. Fig. 1.
The molecular weights obtained are shown in
All three preparations appear to be quite homogeneous, as can be deduced
I
3.8
I
I
I
I
I
I
I
I
I
I--
J
I
[
]
F---
I 600
I
I 800
(I)
3~ 34
32 I
I
~
(2) '0 38 x
~ 35 ~9
~ a4 ~32 o
I
I
[ ~ I F - - -
(3) ~ 38 3.6 54
.i ?"~?,?t-"
32 I 200
[
~
I 400
I
FRINGE DISPLACE M ENT,/z
Figure 1. The weight-average molecular weigbi of v a r i o u s 11 S AChE preparations, plotted against fringe displacement as computed by a computer high-speed equilibrium ultracentrifugation program (4). (1) Acetyleholinesterase A; (2) ace~yloholinesterase C; (3) acetylcholtnesterase B'. All ~ r e e preparations were centrifuged at 10,000 rpm, as described i n t h e text.
from the small variation of the molecular weight plotted vs. fringe displacement (whieh is proportional to concentration) along the ultracentrifuge cell. The preparations have molecular weights in the range of 320,000-350,000, assuming a v of 0. 723
(4).
These values are all significantly higher than the molecular weight of
230,000-260,000
previously reported for AChE preparations with a sedimentation
coefficient of about 11 S (5-7). AChE-B', prepared from extensively autolysed tissue, displays the same specific activity and sedimentation coefficient as AChE-A, AChE-B and AChE-C.
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The molecular weights of the potypeptide components of all four 11 S preparations were determined by performing polyacrylamide gel electrophoresis, in the presence of
SDS and ~-mercaptoethanol.
They all contain two main polypeptide components with
molecular weights of 82,000 + 6,000 and 59,000 ± 4,000, respectively.
However,
whereas in the "native" forms of AChE (18 S + 14 S, Ref. 9) the 82,000 polypepLide
is the major component and the 59,000 polypeptide is present only in small amounts, if at all
(Fig.
2.1,
Ref. 9), ~he relative amounts of the two polypeptides varies
markedly in different 11 S acetylcholinesterase preparations (Fig. 2.2
82 59,000-
and 2.3).
In
Q
25,000-
CI)
C2)
C3)
Fib,ure 2. Acrylamide gel electrophoresis, in the p r e s e n c e of 8DS and ~-mercaptoethanol, of AChE preparations. 50/0 acrylamide gels were used, and stained with Coomassie blue. (1) "Native ' AChE (14 S + 18 SI; (2) AChE~B; (3) AChE-B'.
11 S AChE derived from tissue which had undergone the most extensive autolysis, (AChE-B'), the relative amount of the 82,000 component is reduced and the amount of the 59,000 component is markedly increased (Fig. 2.3).
The increase in the relative
amount of the 59,000 component is accompanied by the appearance of polypeptides of lower molecular weight (~25,000).
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Both the 82,000 and 59,000 components o b s e r v e d b y staining for p r o t e i n contain D F P - b i n d i n g s i t e s (Fig. 3).
Again it can be seen that the r e l a t i v e amount of the
59,000 component, a s r e v e a l e d by D Fl:Lbinding, i n c r e a s e s in the e x t e n s i v e l y a u t o l y s e d 11 S A C h E - B ' (Fig. 3.3).
We found that about 90°/0 of the r a d i o a c t i v e d i i s o p r o p y l -
/ 5
/
,
(2)
• ~59,ooo
~s2,oo%
~0 6 × 4 o_
,000
o~59,000
(3) 50 20~
~82,oooo
0
I0
~ i
20
! 30
40
50
MIGRAT]ON (millimeter)
F i g u r e 3. A c r y l a m i d e gel e l e c t r o p h o r e s i s , in the p r e s e n c e of SDS and ~ - m e r c a p t o ethanol, of 3 H - D F P - l a b e l l e d AChE s a m p l e s . The g e l s were sllced~ r a d i o a c t i v i t y m e a s u r e d and the m o l e c u l a r weigl~s c a l i b r a t e d a s d e s c r i b e d u n d e r Methods. (1) "Native" AChE (14 S + 18 S); (2) AChE B; (3) AChE B ~.
p h o s p h o r y l g r o u p s could be r e m o v e d f r o m boLh components with the s p e c i f i c a c e t y l c h o l i n e s t e r a s e r e a c t i v a t o r 2 - P A M (14) under the conditions d e s c r i b e d by us (8}.
No
r a d i o a c t i v i t y a p p e a r e d to be a s s o c i a t e d w~h the f a s t e r - m o * 4 n g (,~25,000) p o l y p e p t i d e components, indicating that these p o i y p e p t i d e s do not contain active s i t e s . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s in the p r e s e n c e of SDS but in the a b s e n c e of ~ - m e r c a p t o e t h a n o l r e v e a l s that a l l the p r e p a r a t i o n s of 11 S AChE d i s p l a y polypeptides o f 1 6 8 , 0 0 0 4- 8,000 and 88,000 4- 4~000, both of which bind D F P .
Itthus seems
p l a u s i b l e t o a s s u m e that the 160; 000 - !70~ 000 component is a d i m e r i c s t r u c t u r e in which two subunits a r e connected by i n t e r s u b u n ~ disulfide bonds (8).
This p o s s i b i l i t y
was a l s o p r o p o s e d b y F r o e d e and Wilson (15) on the b a s i s of s u c r o s e - g r a d i e n t c e n t r i -
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fugation e x p e r i m e n t s employing 14C-DFP-labet~ed AChE tx,eated e i t h e r with guanidine alone o r with guanidine and ~ - m e r c a p t o e t h a n o L The s e d i m e n t a t i o n eq~i~ibrium and a c r y ! a m i d e gel e l e c t r o p h o r e s i s data~as well as the r e s u l t s of the d e t e r m i n a t i o n s of s values and s p e c i f i c a c t i v i t i e s of the v a r i o u s 11 S AChE p r e p a r a t i o n s ~ can be b e s t explained by a s s u m i n g that a u t o l y s i s can lead to cleavage of the 82~000 peptide chains p r e s e n t in the native enzyme m o l e c u l e , at a l i m i t e d n u m b e r of s u s c e p t i b l e s i t e s .
However, this cleavage does not l e a d to r e l e a s e
of the 59,000 and-.~ 25,000 m o l e c u ) a r weight f r a g m e n t s p r o d u c e d and to d i s r u p t i o n of the q u a t e r n a r y s t r u c t u r e , ur~_~ess the enzyme is denatured.
S e v e r a l g r o u p s have
r e p o r t e d c a s e s in which p r o t e o l y t i c clean,-age p e r s e does not change the o v e r a l l m a s s and q u a t e r n a r y s t r u c t u r e of p r o t e i n s (16~19} and we have a l r e a d y s u g g e s t e d this p o s s i b i l i t y for AChE (2). On the b a s i s of the above, va~fous m o d e l s can be s u g g e s t e d for the q u a t e r n a r y s t r u c t u r e of 11 S AChE. Since ~:native ~r (Le. 18 S + 14 S} p r e p a r a t i o n s of AChE and s o m e 11 S p r e p a r a t i o n s contain r e i a t i v e l y sma}Z amounts of the 80,000 component on SDS e l e c t r o p h o r e s i s in the a b s e n c e of ~-mercaptoethanol0 it s e e m s unlikely that a valid m o d e l will include subunits unl-iffked to any o t h e r sub~mit by disulfide b r i d g e s . We will c o n s i d e r , t h e r e f o r e , on~_y m o d e l s in which p a i r s of subunits a r e linked b y d i s u l f i d e bonds. Two s i m p i e m o d e l s az'e shoa, n in Fig. 4.
In Model_ I the subunits
a r e linked by disulfide bridges~ as aye the two pa~ts of the polypeptide chain which a r e cleaved b y p r o t e o l y s i s o r a u t o I y s i s . The pol-ypeptides a r e a r r a n g e d in p a r a l l e l (i. e. with the s a m e p o l a r i t y f r o m the NH2~terminal to the COOH-terminal residue} as is the c a s e in y - g l o b u l i n (20}.
This m o d e i does not s e e m v e r y s a t i s f a c t o r y , b e c a u s e
w h a t e v e r the p e r c e n t a g e of subunits cleaved e n z y m i c a l l y , one would not e x p e c t a p p e a r a n c e of polypeptides of m o l e c u l a r weight 80,000 on a e r y l a m i d e g e l s i n the a b s e n c e of ~ - m e r c a p t o e t h a n o l , and this is n o t in fact the c a s e (8).
Another s i m p l e
model, Model 12, does not r e q u i r e i n t r a s u b u n i t d i s u l f i d e s and p o s t u l a t e s an a n t i p a r a l l e l a r r a n g e m e n t of polypeptide chains linked by disulfide b r i d g e s in each of which one c y s t e i n e r e s i d u e is beyond the cleavage point.
A n t i p a r a l l e l a r r a n g e m e n t of
polypeptide chains in multisubunit p r o t e i n s h a s indeed been d e s c r i b e d in s e v e r a l e a s e s (21).
This m o d e l is m o r e a t t r a c t i v e s i n c e it can account for the a p p e a r a n c e of
a-,~80,000 s p e c i e s even p r i o r to r e d u c t i o n .
O b v i o u s l y , m o r e complex m o d e l s can
account for the d a t a a s s u m i n g m o r e disulfide b r i d g e s a n d / o r m o r e c l e a v a g e p o i n t s . M o r e o v e r , 11 S AChE is i t s e l f a d e g r a d a t i o n product of "native"'AChE (1,2}, and the q u a t e r n a r y s t r u c t u r e of the "native", f o r m s of the enzyme m u s t take into account i n t e r -
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Vol. 59, No. 1, 1974
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
COOH
COOH
A
k
COOH
A
]I
COOH
NH2
,k
COOH
'1
NH 2
-S--S-
-S--S-
i
-S--S- 1
t NH 2
¢
NH 2
NH2
y
i1'
3~'0-350,000
MW:
COOH
'~I
!
4TM
:y - S - - S - ¢,I
-S--S- i
MW:
NH2
COOH
NH 2
320-350,000
~Hz
C00H
A,
-S--S-
J I
COOH
NH 2
COOH
,k
COOH
.H~ C~OH
i -s-s-Ck
,b_ -s
-S--S-
¢1 NH 2
MW:
-S-S-
1"
Y COOH
NH2
~165~000 COOH
.s
80,000
COOH
.I,
NH2
MW: COOH
C00H
SH
qM'
HS-
'~#v
HS -
HS
~ SH
I}
- S H HS -
4 ..
,I' NH2
MW:
HS-
l' NH2
¢ NH2
~ eo,ooo; ~6o,ooo;~ 25000
MW:
I~
-SH
NH 2
~ 8o,ooo;~6o,0oo,~ 25,000
Figure 4. Schematic models for the subunit structure of 11 S AChE~ together with the products to be expected under the conditions employed for SDS-acrylamide gel electrophoresis, both in the presence t~nd absence of [~-mercaptoethanol, subsequent to cleavage of either one or both polypeptides of the dimer. The site where a cleavage can occur i s indicated by a serrated line. For discussion s e e text.
actions between larger numbers of subunits, and also Lhe presumably specific mode of attachment of the "tail" of the native enzyme to the multisubunit head (3,4).
However,
any model should take into account the fact that reducing agents are n e c e s s a r y for complete dissociation of the AChE molecule into its subunits, and for r e l e a s e of fragments from the cleaved subunits, subsequent to proteolysis or autolysis. Thus our results indicate that 11 S AChE is a tetramer of molecular weight 320,000-350,000, each subunit being of about 80,000.
Our observation that the
principal subtmits present in electric eel AChE have molecular weights of about 80,000 and 60,000 is in agreement with recent results from other laboratories (10-12). Our r e s u l t s also indicate that p01ypeptides of about 60,000 and 25,000 appear gradually following autolysis or tryptic digestion, but that these polypeptide fragments, formed by cleavage of the 80,000 subunit, are not released from the molecule unless it is denatured.
Similar conclusions have r e c e n t l y been reached by Chen et al. (22) on the
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
basis of acrylamide gel electrophoresis and exclusion chromatography in 6 M guanidine solutions. Leuzinger et al. (6) reported earlier that 11 S AChE is a t e t r a m e r of 260,000, containing four 64,000 subunits.
We cannot exclude the possibility that under certain
conditions cleavage of the 80,000 polypeptide is followed by r e l e a s e of polypeptide fragments, even p r i o r to denaturation. Such a process could result in formation of an active AChE t e t r a m e r of 230,000-260,000, as reported previously (5-7), with subunits of 64,000 or less (6,7).
However, we did not observe the formation of such a species
in our preparations.
Acknowledgments The support of the Volkswagen Foundation is gratefully acknowledged. We wish to thank Mrs. Esther Saksel-Roth for her skilful technical assistance and Drs. Martin Hirshfeld and Robert Josephs for help with the ultracentrifuge experiments. REFERENCES
1. 2. 3.
Massouli~, J . , and Rieger, F. (1969} Eur. J. Biochem. 1__~1,441-455. Dudai, Y., Silman, I., Kalderon, N., and Blumberg, S. (1972) Biochim. Biophys. Acta, 268, 138-157. ./ Rieger, F . , Bon, S., Massouhe, J., and Cartaud, J. (1973) Eur. J. Biochem.
3~, 539-547. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Dudai,Y., Herzberg, M., and Silman, I. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2473-2476. Kremzner, L.T., andWilson, I.B. (1964) Biochemistry, 3, 1902-1905. Leuzinger, W., Goldberg, M., and Cauvin, E. (1969) J. Mol. Biol. 40, 217-225. Millar, D.B., and Grafius, M.A. (1970) FEBS Lett. 1._22, 61-64. Dudai,Y., and Silman, I. (1971) FEBS Lett° 1_6_6,324-328. Dudai,Y., Silman, I., Shinitzky, M., and Blumberg~ S. (1972) Proc. Natl. Aead. Sci. U.S.A. 6__99,2400-2403. Berman, J.D. (1973) Biochemistry, 1_22, 1710-1715; Bock, E., Chen, Y. T . , and Rosenberry, T . L . (1973) Fed. Proc. 322, 510. Powell, J . T . , Bon, S,, Rieger, F . , and Massoul~d~ J. (1973) FEBS Lett. 36, 17-21. Rothenberg, M . A . , and Nachmansohn, D. (1947) J. Biol. Chem. 168, 223-231. Wilson, I . B . , Ginsburg, S . , and Quan, C. (1958) Arch. Biochem. Biophys. 7._7_7, 286-296. Froede, H . C . , and Wilson, I.B. (1970) Israel J. Med. Sci. 6, 179-184. Ullmann, A . , Jacob, F . , and Monod, J. (1968) J° MoL Biol. 3.~2~ 1-13. Goldberg, M.E. (1969) J. Mol. Biol. 4._66, 441-446. Labeyrie, F . , andBaudras, A. (1972) Eur. J. Biochem. 2_55, 33-40. Jacq, C., and Lederer, F. (1972) Eur. J. Biochem° 2.~5~41-48. Gall, W . E . , Cunningham, B . A . , Waxdal, M . J . , Konigsberg, W . H . , and Edelman, G.M. (1968) Biochemistry, 7, 1973-1982. Matthews, B.W., and Bernard, S,A. (1973) Ann. Rev. Biophys. Bioeng. 2, 257-312. Chen, Y. T., Rosenberry, T . L . , and Bock, E. (1974} manuscript in preparation.
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