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Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain
Neurochemistry International, Vol. 3, No. 5, pp, 311-321, 1981 Printed in Great Britain.
0197-0186/81/050311-11 $02.00/0 © 1981 Pergamon Press Ltd.
MOLECULAR FORMS OF ACETYLCHOLINESTERASE PRESENT IN THE WHITE AND GREY MATTER OF PIG BRAIN MARGARETS. Y. CHAI, CHARLESA. REAVILL*,CECELIOJ. VIDAL~ and DAVID T. PLUMMER Department of Biochemistry, Chelsea College, University of London, Manresa Road, London SW3 6LX, U.K.
(Received 16 March 1981; accepted 13 July 1981) Abstract--Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove aeetyicholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter. The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter. Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge. Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed. A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.
Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7.) has been found in the central nervous system of all vertebrates studied, although the distribution of the enzyme in the brain is very uneven (Silver, 1979). The AChE in mammalian brain is known to exist in multiple molecular forms which have been identified by a variety of sedimentation, chromatographic and electrophoretic techniques. The multiple forms appear to differ in size, since gel filtration has revealed the presence of a number of species with a range of molecular weights (Chan et al., 1972; Wenthold et al., 1974; Gordon et al., 1976). This has been confirmed
by centrifugation studies which showed the enzyme to exist in forms with different sedimentation constants (Rieger and Vigny, 1976). The molecular forms of brain acetylcholinesterase also seem to differ in terms of their electrical charge as revealed by several electrophoretic techniques (Davis and Agranoff, 1968; Srinivason et al., 1972; Vijayan and Brownson, 1974). The existence of multiple molecular forms of acetylcholinesterase is thus well documented, but most workers have used the whole brain or the cortical region for their enzyme preparations, and there is little information on the properties of acetylcholinesterase and the molecular species present in different Present addresses: * Department of Neurology, Institute regions of the brain. For example, studies in our own laboratory have been carried out on pig cerebral corof Psychiatry Denmark Hill, London SE5, U.K. tex (Mclntosh and Plummer, 1973; Plummer et al., t Departmento Interfacultativo de Bioquinica, Facuitad de Medicina, Espinardo-Murcia, Spain. 1975) and subcellular fractions of this tissue (Mcln311
312
MARGAREI S. Y. CHM et al.
tosh a n d P l u m m e t , 1976) but o t h e r regions of pig brain have n o t been examined. This work has n o w been e x t e n d e d to a study of A C h E in the white m a t t e r o f pig brain and the results are c o m p a r e d with the e n z y m e o b t a i n e d from the grey matter. T h e study o f acetylcholinesterase from the two parts of the brain should therefore p r o v i d e a crude but nevertheless useful c o m p a r i s o n o f axonal (white matter) a n d synaptic (grey matter) acetylcholinesterase.
EXPERIMENTAL Materials Porcine brains were obtained from the Co-operative Society Slaughter House, Woolwich, London. Potato starch was supplied by BDH, Poole, Dorset and fine chemicals were purchased from Sigma, London. The acetylcholinesterase inhibitor BW 284 c51 was obtained from Burroughs Wellcome, Beckenham, Kent. Ethopropazinc was supplied as a gift from May & Baker Ltd. All solutions were prepared in distilled water, redistilled from an all glass still. Assay of acetylcholinesterase The activity of acetylcholinesterase was determined at 30°C and pH 7.9 using a pH-stat (Mclntosh and Plummer, 1976). A colorimetric method was employed when a large number of fractions had to be assayed as in starch-block electrophoresis and density gradient centrifugation. In this case, the substrate used was acetylthiocholine and the mercaptide formed from the hydrolysed thioester was reacted with an oxidizing agent 5,5'dithio-(bis)-(2-nitrobenzoic acid), DTNB to give a product that absorbs at 412 nm (Ellman et al., 1961). Protein assay The protein content of a membrane suspension was measured by a modified biuret method where 1.5~o w/v sodium deoxycholate was incorporated into the reagent to solubilize all of the proteins present. The biuret reagent (2ml) was added to the protein solution (1 ml) and the mixture heated on a boiling water bath for 1 rain. At the end of this period, the mixture was cooled to room temperature and the extinction measured at 540 nm. The method of Lowry et al. (1951) was used when low concentrations of protein were present. The detergent Triton X-100 interferes with this assay, but this was overcome by centrifuging the precipitate (10000, 5min) and incorporating the detergent in the reagent blank and standards (Hartree, 1972; Chandarajan and Klein, 1975). Solubilization of the enzyme All solubilization procedures were performed on porcine brains obtained on the day of use from the slaughter house. The membranes and blood vessels were removed from the surface of the brains and the white and grey matter separated and dispersed in a Waring Blender for 5 rain at 4°C with 30 mM sodium phosphate buffer (pH 7.0) to produce a 10~o homogenate (g wet weight per ml). Centrifugations were then carried out on an MSE SS 50 preparative ultracentrifuge using an 8 × 50ml or a 10 × 10ml capacity
rotor. The enzyme was considered to be soluble if it remained in the supernatant after being centrifuged al 100,000 o for 1 h. The supernatant from this extraction is known as the 'naturally soluble' form of the enzymc~ Further solubilization of acetylcholinesterase was carried out by resuspending the pellet in an appropriate medium and gently stirring. The extract was then centrifuged at 100,000 0 for 1 h and the supernatant assayed for AChE activity. Triton X-100, lysolecithin and EDTA were prepared in 30raM sodium phosphate, pH 7 while sodium cholate and sodium deoxycholate were dissolved in 50 mM Tris buffer pH 7.5. The condilions varied slightly according to the solubilizing agent employed and 30 min at 37 C was found to be optimal for the naturally occurring detergents, sodium cholate, sodium deoxycholate and lysolecithin (Marples et al., 1959; McArdle et al., 1960). The synthetic detergent Triton X-100 (Ho and Ellman, 1969) needed only 10rain at room temperature, while EDTA extraction (Chan et al., 1972; Hollunger and Niklasson, 1973) required stirring for 2 h at 4 C . In the latter case, the supernatant was removed and the pellet treated twice more in the same way. The concentration of the reagents used are given in Fig. 1. A~hnity ehromatooraphy The AChE was purified on an affinity column of Sepharose 4B linked to the ligand (1-methyl-9-(Na-~-aminohexanoyl)-fl-aminopropylamino)-acridinium bromide hydrobromide (Dudai and Silman, 1974). The preparation of this column (MAC-agarose) and the method used was the same as that previously described for the purification of AChE from pig cerebral cortex (Reavill and Plummer, 1978). Detergent was present throughout the preparation and the purified enzyme contained 0.1°/,, w/v Triton X-100. Starch-block electrophoresis Several of the enzyme preparations were subjected to electrophoresis on starch grains (Plummer, 1978). Potato starch was washed four times by decantation; twice with water and twice with the electrophoresis buffer (sodium phosphate, 0.1 M, pH 7). In some experiments, Triton X-100 was incorporated into the buffer at a final concentration of 1°/,~ w/v. Excess buffer was decanted and the starch pressed into a block with the aid of a Perspex former. Wicks consisting of several layers of muslin were used to connect the block with the buffer in the electrode tanks (0.1 M, sodium phosphate pH 7). The power pack was then connected and the block equilibrated at 6 V cm-for I h at 4°C. The enzyme sample containing 5-10 mg of protein was then applied to a laterally cut groove, 0.5 cm wide towards the cathode end of the block and bovine serum albumin stained with bromphenol blue was applied as a marker in a cavity in line with the groove. The cavity and groove were filled with starch and electrophoresis carried out at 6 - 7 V c m -~ for 18h. The position of the markers was carefully noted and the longitudinal section containing them discarded. The remainder of the block was cut transversely into strips 0.5 or 1 cm wide, placed in a sintered glass funnel and the enzyme elute~l with 2 ml aliquots of buffer. The acetylcholinesterase in each fraction was then assayed by the EIlman method.
Acetylcholinesterase in pig brain
Polyacrylamide-oel-electrophoresis Multiple forms of acetylcholinesterase were separated on the basis of differences in size by prolonged electrophoresis on a concave gradient of polyacrylamide (Margolis and Kenrick, 1968; McIntosh and Plummer, 1973). The gels were then stained for enzyme activity with acetylthiocholine as substrate by the method of Koelle (1951) as modified by Lewis and Shute (1966).
Density gradient centrifugation Enzyme samples were placed on top of a linear sucrose gradient (5 to 60~o w/v) prepared in 5 ml polycarbonate tubes then centrifuged overnight (17h) at 100,000g. The standard protein bovine catalase with a sedimentation coefficient of 11.4 S and a molecular weight of 240,000 was mixed with the enzyme sample to act as a convenient marker. At the end of the centrifugation, the tubes were punctured with an M.S.E. tube piercer and fractions collected and assayed for acetylcholinesterase activity. Catalase was assayed by following the decrease in extinction at 240 nm of a mixture containing 3 ml sodium phosphate buffer (10 mM, pH 7.5), 20 #1 of hydrogen peroxide (0.9 M) and 20/tl of the fraction. Activities were calculated in terms of the change in absorbance per minute and the fraction with maximum catalase activity was noted. The efficacy of the technique was tested by running standard proteins on the gradients and checking that a linear response was obtained when the sedimentation constants were plotted against the distance migrated down the tube (Martin and Ames, 1961). The sedimentation constant of each peak of enzyme activity was calculated from the distance travelled down the tube relative to the catalase marker (Martin and Ames, 1961). A crude estimate of the molecular weight of each species was obtained from the formula: $1/$2 = (MWI/MW2) 2/3 when $1 = sedimentation constant of unknown, $2 = sedimentation constant of catalase (11.4S), MW1 = molecular weight of unknown and MW2 = molecular weight of catalase (240,000). RESULTS
Solubilization of acetylcholinesterase from white matter Acetylcholinesterase is a membrane-bound enzyme (Nachmansohn, 1971) and so the initial experiments involved the extraction of the white matter with a variety of agents in an attempt to bring the enzyme into solution. The AChE activity in the uncentrifuged homogenate is taken to be 100~ and the enzyme was considered to be soluble if it remained in the supernatant after centrifugation at 100,000 g for 1 h. Dilute buffer solution. Extraction of white matter with water or dilute buffer (30 mM sodium phosphate, pH 7.0) solubilized from 13 to 179/o of the total activity. This fraction was referred to as the 'naturally soluble' enzyme and subsequent extraction of the pellet did not bring any more enzyme into solution. Attempts were then made to solubilize the membrane-bound enzyme by extracting the pellet with a variety of media.
313
EDTA. Repeated homogenization and incubation with 3 0 m M sodium phosphate buffer containing 1 m M EDTA brought 39/0 of the enzyme into solution (range 2.8-3.29/0). The yield was not improved by altering the concentration of the brain homogenate. Triton X-IO0. The non-ionic detergent Triton X-100 was not very effective as a solubilizing agent and as the total Triton X-100 concentration was raised from 0.1 to 2~o w/v, the amount of AChE solubilized increased from 16 to only 18~o of the total homogenate activity. Lysolecithin. Lysolecithin was a more effective solubilizing agent than EDTA or Triton X-100 and when the 100,000 g pellet was extracted with 12 m M lysolecithin, about 33~o (range 30-36~o) of the total homogenate activity was solubilized from the pellet. Bile salts. Bile salts were then used in an attempt to bring the enzyme into solution and the early results with sodium cholate were disappointing. Extraction of the 100,000g pellet with 1~o w/v sodium cholate solubilized only 18.5~o (range 16.5-20.59/o) of the AChE activity. However, sodium deoxycholate was very effective as a solubilizing agent and a 0.2~o w/v concentration of the detergent brought 699/0 (range 66-72~o) of the total homogenate activity into solution. Increasing the concentration of sodium deoxycholate, solubilized more of the enzyme with a maximum yield of 88~o (range 87-89~o) using 0.3~ w/v sodium deoxycholate. Therefore all of the AChE activity of the homogenate of white matter could be accounted for with 15~o extracted with dilute buffer and 88~ with 0.3~o w/v sodium deoxycholate. However, although concentrations of detergent greater than 0.2~o w/v solubilized more of the enzyme, the problem of lipid coming out of solution made it impracticable for routine use so that extraction with 0.2~o w/v sodium deoxycholate was adopted as the standard procedure for white matter. There was essentially no difference in the AChE activity between a 10~o w/v suspension of the white matter of porcine brain in dilute buffer and a 10~o w/v suspension of brain in any of the solubilizing agents used, indicating the absence of activation or inhibition. A summary of these results and a comparison with the grey matter of brain is shown in Fig. 1 and Table 1. Specificity of the enzyme preparations The substrate specificities of the sodium deoxycholate solubilized acetylcholinesterase and the naturally soluble enzyme were examined by measuring the rates of hydrolysis of a number of substrates and compar-
314
MARGARE]
%
0 A c t i v i t y in IO0,OOg
al.
S. Y. C H A I e t
Total octiv,ty solubilized from the IO0, O00g pellet 20
40
60
80
pellet
IC,C~ I
E xtroction method
I
EDTA (traM}
]
Triton X - I00 (1% w/v)
]
Lysotecithin (12 mM) Sodium eholote (1% w/v) Sodium deoxycholote (0.2 % w/v)
Fig. 1. The solubilization of membrane-bound acetylcholinesterase from the white (11) and grey matter (E]) of porcine brain. The total enzyme activity in the homogenate is shown as 100~o which is made up of 15°/o that can be extracted with dilute buffer and 85,% which is membrane-bound ( . . . . . . . ). The amount of enzyme solubilized is that which remains in the supernatant after centrifugation at 100,000 g for 1 h. Details of the extraction procedures are given in the text. ing them with acetylcholine. The results obtained show that the 'naturally soluble' and the detergent solubilized preparations had very similar specificities (Table 2). Data obtained for the acetylcholinesterase in porcine grey matter are also included for comparison. The Michaelis constants (K,,) were measured for the crude homogenate, the naturally soluble and the detergent solubilized forms of the enzyme from pig brain and the results are shown in Table 1. In the case of the white matter, the naturally soluble form of the enzyme had the highest K,, with the homogenate significantly lower than this and the detergent solubilized enzyme slightly less than the homogenate value.
The Michaelis constants for the grey matter were the same as those for the corresponding preparations from the white matter of porcine brain.
Purification by affinity chromatography The deoxycholate solubilized preparation (100150 ml) was applied to the affinity column at a rate of about 3 0 m l h -1. Following this, the column was washed with the elution buffer (30 raM, sodium phosphate, pH (7.0) until no protein could be detected in the eluate, The acetylcholinesterase inhibitor, decamethonium bromide ( l O m M ) w a s then incorporated into the buffer and the column washed until no further enzyme could be detected in the fractions. The
Table 1. Enzyme activity and Michaelis constants of AChE prepared from the white and grey matter of pig brain
Preparation
Acetylcholinesterase activity (•moles min- 1) per g wet weight (o(,) per mg protein
Michaelis constants (pM), acetylcholine
Homogenate White matter Grey matter
3.80 5.11
(100) (100)
0.048 0.082
70 76
Naturally soluble enzyme White matter Grey matter
0.58 0.71
(15) (14)
0.030 0.028
106 118
Detergent solubilized enzyme White matter Grey matter
2.60 2.85
(69) (56)
0.062 0.142
64 62
The values shown are the mean of 4-6 experiments.
315
Acetylcholinesterase in pig brain 0.6
I
NaCI
Decamethoniurn bromide (10 mM) /
Z.
E E
0.~
_=
E rr
=L
E
0.2
E
c~
t3
E LU 120
240
Elution volume,
5bO
rnL
Fig. 2. Elution profile of acetylcholinesterase from a column of MAC-agarose. The elution medium used was 30 mM sodium phosphate buffer, pH 7.0 and the arrows mark the point at which the elution medium was changed to include the reagents shown. Experimental details are as in the text. eluting medium was then changed to sodium phosphate buffer containing 1 M NaCI when a further peak of enzyme activity was obtained. The fractions containing the enzyme were dialysed (Reavill and Piummer, 1978) and the activity deter-
mined. The elution profile is shown in Fig. 2 and the AChE activity of the peaks in Table 3. The total yield of the enzyme from the column was 35~o with 24~o of the enzyme activity eluted in the decamethonium fraction (peak I) and 11~o of the activity eluted by 1 M
Table 2. The relative hydrolysis of choline esters by brain acetylcholinesterase
Buffer soluble enzyme
Substrate Acetylcholine iodide
Activity as % of acetylcholine iodide White matter Grey matter Detergent soluble Detergent soluble enzyme Buffer enzyme (0.2% w/v sodium) soluble (1% w/v (deoxycholate) enzyme Triton X-100)
100
Propionylcholine iodide Butyrylcholine iodide Acetyl=fl-methylcholine bromide Tributyrin
100
100
82.4
80.9
2.0
100
83.0
84.0
1.8
0
0
21.3
22.0
20.0
22.0
7.4
8.0
4.0
5.0
The substrate concentration in all cases was 1 mM and the activity was determined with a pH-stat as described in the text. The rate of hydrolysis is expressed as a percentage of that found with acetylcholine iodide. The values shown are the average of 4 experiments. Table 3. The purification of acetylcholinesterase from the white matter of pig brain on an affinity column of MAC-agarose
Total applied
Protein (mg)
Enzyme activity (#)
Specific activity (# mg- t protein)
Yield (%)
Purification (times)
629.0
39.84
0.063
--
--
Recovered in peak I
0.52
9.69
18.63
24.0
300
Recovered in peak II
0.36
4.55
12.64
11.0
200
The units of enzyme activity (#) represent ~moles acetylthiocholine iodide hydrolysed per minute.
316
M A R G A R E T S. Y . C H A I e l
020 JA2
(a} Deoxycholote - solubilized enzyme
L E Ol5 'c E ::::L
010
>~ > 005
~ >.
0
I
1
ezm;
(b) Naturelly soluble
0 05[
0
I
5
~o
Distance from anode,
115
I 2o
cm
Fig. 3. Starch-block electrophoresis of acetylcholinesterase from the white matter of porcine brain. (a) Deoxycholate solubilized enzyme. (b) Naturally soluble enzyme. The arrow .L denotes the point of application of the sample while Alb marks the position of the albumin marker. Experimental details as in the text. NaC1 in the second peak (II). The purification of the acetylcholinesterase in peak I was 300 fold while that in Peak II was 200 fold. The enzyme in the first peak was therefore used in subsequent investigations.
Electrophoresis Starch-block electrophoresis. The naturally soluble acetylcholinesterase from the white matter showed one peak of enzyme activity and the deoxycholate solubilized enzyme also gave only one peak of enzyme activity with a similar electrophoretic mobility to that of the soluble enzyme (Fig. 3). In contrast to this, the Triton solubilized enzyme from grey matter gave a very broad band of activity with a low electrophoretic mobility (Fig. 4a). However, if 1% w/v Triton X-100 was incorporated into the block and buffer system, then two peaks of activity were obtained, which was the same as the pattern obtained for the naturally soluble preparation from grey matter (Fig. 4b). The fast moving peaks from all the preparations showed a range of molecular weights during electrophoresis on a gradient of polyacrylamide, whereas the slow moving peaks present in the grey matter appeared to be
~ll.
an aggregated form of the enzyme with a high molecular weight. This is because peak 11 from the starchblock barely entered the gradient gels giving an intense surface staining and also migrated to the bottom of the centrifuge tube during density gradient centrifugation. The purified enzymes showed the same characteristics as the detergent solubilized enzymes when subjected to electrophoresis on a starch-block. Gradient polyacrylamide-gel electrophoresis. Electrophoresis on a gradient of polyacrylamide showed multiple bands of enzyme activity in all the enzyme preparations examined. The results are shown in Fig. 5 where the approximate molecular weights are indicated on the assumption that the acetylcholinesterase molecules are spherical. The naturally soluble enzyme from the white matter had two major bands of activity with molecular weights of 660,000 and 260,000 a less intense but broad band with a molecular weight of 120,000 and a faintly staining band at 400,000. The sodium deoxycholate solubilized enzyme had three bands of activity in common with the naturally soluble acetylcholinesterase (400,000, 260,000, 120,000) and also a species
AIb
]I
-EL(O) Triton - solubilized enzyme
004 L E
[[
E
0 02
r \
o E
>
c
0
04
(b) Naturally soluble
enzyme
002
0
F
l/<
I0
Distonce from onode,
20
30
cm
Fig. 4. Starch-block electrophoresis of acetylcholinesterase from the grey matter of porcine brain. (a) Triton solubilized enzyme. (b) Naturally soluble enzyme. Other details as in Fig. 3.
Acetylcholinesterase in pig brain Detergent solubilized
Noturolly soluble w
W
G
G
317
Purified enzyme W
G
Molecular weiqhts II
I
I
I I
-
-[] i
• I
- -
i
[]
- -
--
-
I
-
i
-
660,000
400, 000 z6o, ooo 180, o o o 130, 000
68,000
I
m
Fig. 5. Electrophoresis of acetylcholinesterase preparations from porcine brain on a gradient of polyacrylamide. W denotes white matter and G the grey matter. Depth of staining: strong I ; moderate l ; light m. Experimental details as in the text. with a MW of 180,000. The peaks of activity from the starch-block electrophoresis of both enzyme preparations gave the same staining patterns as those shown in Fig. 5. The staining pattern for the purified enzymes was very similar to the deoxycholate extract, but the bands were sharper and more intensely stained than in the other preparations. There were, however, some minor differences in that the faint band at 400,000 disappeared on purification while the broad band with an average molecular weight of 120,000 was resolved into two species with molecular weights of 115,000 and 130,000. The results for the grey matter are also shown in Fig. 5 and the differences and similarities to the patterns obtained for the white matter preparations will be considered in the discussion. When 10pM eserine was incorporated into the staining system, no AChE activity was detected showing that the bands of activity were due to cholinesterase. If the acetylthiocholine iodide was substituted by butyrylthiocholine iodide, a few faint bands of activity were seen, but none of them corresponded to the bands shown in Fig. 5. The activity detected therefore appears to be due to acetylcholinesterase rather than butyrlcholinesterase. Finally, the incorporation of 30 pM ethopropazine into the staining system had no effect on the bands produced when acetylthiocholine was used as the substrate confirming that the enzyme detected was acetylcholinesterase.
Density gradient centrifugation White matter. The naturally soluble form of the
enzyme gave one peak of activity with a sedimentation constant of 9.5 S (Fig. 6b). The results for the deoxycholate solubilized enzyme was very similar with one peak at 10.5 S provided that sodium deoxycholate was incorporated into the sucrose (Fig. 6a). If the detergent was omitted, then the AChE sedimented to the bottom of the tube indicating the presence of a high molecular weight species. In view of this, detergent was always present during centrifugation of the deoxycholate-solubilized enzyme. The peaks of activity obtained from the starchblock electrophoresis of both enzyme preparations also gave only one peak of activity with a sedimentation coefficient of about 11 S. The purified enzyme when centrifuged on a sucrose gradient was found to have the same sedimentation constant as the crude enzyme solubilized with 0.2~o w/v sodium deoxycholate. Grey matter. When fresh preparations of Triton solubilized AChE were centrifuged on sucrose gradients, one major peak of activity was obtained corresponding to 11-12S. The naturally soluble acetylcholinesterase consistently showed a major peak of activity at 11-12 S, and there was also a suggestion of some minor shoulders indicating the presence of higher molecular weight species, but the resolution was insufficient to enable sedimentation constants to be assigned to them with any degree of accuracy. The peaks of the acetylcholinesterase preparations obtained by starch-block electrophoresis were also subjected to density gradient centrifugation. In all cases, peak II migrated rapidly to the bottom of the gradient indicating a high molecular weight species
318
MARGARI-TS. Y. CHAIet al. Catalase 1145 0to-
(a} Deoxycholate-solubilized
enzyme
E g c o
g o c +-
005
•
/
2 to
./
,
~
.e/
,, e . , ~
c
o= o >"
~ Catolase 11.45
005
t~
"~
0
q
(b) Naturally soluble
enzyme
2 3 4 Fraction, mt
5
Fig. 6. Sucrose density gradient centrifugation of acetylcholinesterase prepared from the white matter of porcine brain. I denotes the position of the catalase marker (l 1.4 S). The top of the tube is on the left of the diagram so the sedimentation direction is as shown (--*). Experimental details as in the text. greater than 1 x 106. Peak 1 on the other hand showed a major band of activity at 11-12 S. Acetylcholinesterase purified by affinity chromatography was also centrifuged on a sucrose gradient and was found to have the same sedimentation characteristics as the crude Triton solubilized enzyme both before and after starch-block electrophoresis. DISCUSSION Acetylcholinesterase is present in the white matter of porcine brain in significant quantities, although the activity per g wet weight or per mg protein is less than that found in the grey matter (Table 1). Extraction of the white matter of pig brain with dilute buffer solution or water solubilized 15~ of the total acetyicholinesterase activity present in the tissue. This value for the 'naturally soluble' form of the enzyme is very similar to that obtained by other
workers using other sources of the enzyme (Ho and Ellman, 1969; Devonshire, 1975; Oderfeld-Nowak and Skangiel-Kramska, t976: McIntosh and Plummer, 1973). The soluble form of the enzyme does not appear to be in equilibrium with the membrane enzyme, since no more enzyme was brought into solution when the 100,000 0' pellet was re-extracted with buffer. Identical results were obtained for the grey matter so the relative amount of soluble AChE and its specific activity appears to be the same in both regions of the brain (Table 1) although its precise function is not known. Several attempts were then made to bring the membrane-bound form of the enzyme into solution by extracting the 100,000 0 pellet with a variety of agents. Ethylenediaminetetraacetic acid (EDTAt is known to facilitate the removal of proteins from membranes probably by disrupting the divalent ion bridges which stabilize protein-lipid complexes (Hollunger and Niklasson, 1973; Maddy and Dunn, 1973). Several groups have, therefore, used repeated extraction with EDTA to solubilize AChE and obtained up to 70}o of the enzyme in a soluble form (Chan et al., 1972; Hollunger and Niklasson, 1973: Wenthold et al., 1974) but in the case of the white matter, only 3% could be solubilized with EDTA. The association between AChE and the membrane seems to be quite strong and so detergents were used as these are usually successful at solubilizing proteins, which are firmly bound to the lipid matrix of membranes (Helenius and Simons, 1975). Extraction with the non-ionic detergent Triton X-100 was not very effective, since only 17"~i of the enzyme activity could be extracted with a concentration of 1~,,;w/v. This is in marked contrast to the 85}/o solubilization obtained for AChE from rat brain (Ho and Ellman, 1969). Lysolecithin was better than Triton X-100 as a solubilizing agent and extraction with a 12 mM concentration of the detergent resulted in the release of 33}0 of the total homogenate activity from the pellet. The yield, however, was still rather low, so for this reason and the expense, lysolecithin was not used as a routine solubilizing agent. Bile salts have been successfully used to solubilize many membrane proteins without loss of biological activity (Spatz and Strittmatter, 1971; Meunier et al., 1972; Snary et al, 1974) and since they are stronger detergents than Triton X-100 or lysolecithin it was felt that cholate or deoxycholate might give a good yield of soluble ACHE. Extraction with 1~ w/v sodium cholate was disappointing, with a yield of 19~o of the enzyme activity, virtually the same as that obtained with Triton X-100. However, the results with sodium
Acetylcholinesterase in pig brain deoxycholate were very satisfactory and a 0.3~o w/v solution of the bile salt solubilized all of the AChE activity in the pellet. Unfortunately, lipid material came out of solution at this concentration which made it difficult to remove the supernatant and so a concentration of 0.2 w/v deoxycholate was routinely used which avoided this problem. The results summarized in Fig. 1 show that when the tissue was extracted with EDTA, lysolecithin or Triton X-100, much lower yields were obtained for the white matter than the grey. Furthermore, the strong detergent sodium deoxycholate was needed to solubilize the AChE in white matter whereas the mild detergent Triton X-100 which was satisfactory for the grey matter was unable to dissociate the enzyme from the membrane in sufficient yield. These results suggest that the AChE in the white matter of porcine brain is more firmly bound to the membranes than the enzyme in the grey matter. Acetylcholinesterase was purified by affinity chromatography on a column of MAC-agarose that was originally developed by Dudai and Silman (1974) for the purification of AChE ~'rom electric eel. The AChE was firmly bound to the column and could be eluted with 10 mM decamethonium bromide (Fig. 2). The yield from this peak (I) was 24~o and the specific activity was 18.63 gmoles min- 1 mg protein- 1, a purification of about 300 fold (Table 3). When the Triton solubilized enzyme from grey matter was purified on the MAC-agarose column, the yield of enzyme in the decamethonium peak was 44yo with a specific activity of 147.6, a 900 fold purification (Reavill and Plummer, 1978). A second peak of enzyme activity (II) was eluted from the column with M NaC1 and accounted for a further 11~o of the enzyme activity. The grey matter also showed a second peak following elution with M NaC1 amounting to 13~o of the enzyme activity (Reavill and Plummer, 1978). In both cases, some binding to the column would seem to occur by electrostatic rather than biospecific interactions indicating a similarity in the two preparations. However, there are significant differences between the binding of the AChE from the white and grey matter to the column which could reflect the differences in the binding of the enzyme to membranes in vivo. The binding characteristics of the AChE from the white and grey matter may be different, but the kinetic properties of the enzyme from the two sources appear to be the same. The Michaelis constants (K,) for a particular preparation are in the same range (Table 1) and are similar to the values obtained from other species (80-140#M). (Jackson and Aprison, 1966; Ho and Ellman, 1969; Chan et al., 1972). The
319
specificity of the preparations from white and grey matter are also very similar and are typical of acetylcholinesterase (Table 2). Starch-block electrophoresis was used as a semipreparative separation of molecular forms which differ in charge. The deoxycholate solubilized AChE and the naturally soluble enzyme both gave a single peak on electrophoresis. However, the naturally soluble enzyme appears to have a slightly higher negative mobility than the bile salt solubilized enzyme (Fig. 3). The purified enzyme showed the same characteristics as the deoxycholate extract. In the case of the grey matter, two peaks of activity were obtained for the naturally soluble form of the enzyme with a fast migrating peak (I) as the major species (Fig. 4b). The same pattern was obtained for the membrane enzyme after it had been solubilized with Triton X-100 provided that detergent was present in the electrophoresis block and buffer. However, if the detergent was excluded from the block then a zone of activity was obtained corresponding to peak II (Fig. 4a). In all cases, peak II sedimented to the bottom of the tube during density gradient centrifugation and barely penetrated the polyacrylamide gel during electrophoresis (Fig. 5). This suggests that the molecular weight of the AChE in peak II is very high and is probably an aggregate of acetylcholinesterase molecules. In contrast to this, sedimentation and electrophoretic measurements clearly showed that peak I was not an aggregated form of the enzyme. These results show that both the naturally soluble and the Triton solubilized forms of the enzyme from grey matter have a tendency to aggregate and that this is most marked in the case of the membrane enzyme soluzilized with Triton. In the absence of Triton X-100, virtually complete aggregation occurs (Fig. 4a) so that detergent is necessary to keep the enzyme extracted from the membrane in solution. The hydrophic nature of some of the molecules of acetylcholinesterase has also been seen in other preparations (Ho and Ellman, 1969; Ott and Brodbeck, 1978) and discussed in recent reviews (Massoulie, 1980; Massoulie et al., 1980). In this present study the aggregated acetylcholinesterase (peak II) would not enter polyacrylamide gel even in the presence of lYo w/v Triton X-100 so it appears that the aggregation could not be reversed by detergent and may well be irreversible as suggested by Hollunger and Niklasson (1973). All four preparations of AChE showed only one unaggregated form of the enzyme (Figs. 3 and 4) with slight difference in the electrophoretic mobility for the enzyme from the white and the grey matter. The
320
MARGARETS. Y. (THAIet al.
single peak on electrophoresis shows the presence of only one charged entity and suggests that the molecular species of brain AChE do not arise from difference in charge. Centrifugation on a sucrose gradient was therefore used to see if AChE exists in multiple forms of different molecular weights. The naturally soluble AChE from white matter gave a single peak with sedimentation constant of 9.5 S whereas the deoxycholate solubilized enzyme migrated to the bottom of the tube indicating the presence of a highly aggregated species. However, if 0.2% w/v sodium deoxycholate was incorporated into the sucrose gradient, the aggregation was abolished and the enzyme resolved into a single peak of activity with a sedimentation constant of 10.5 S (Fig. 6). An identical result was obtained for the purified enzyme both before and after starch-block electrophoresis. Very similar results were obtained from the preparations from grey matter with a peak of 11 12 S. As with the white matter, detergent had to be present in the sucrose gradient to prevent aggregation of the Triton solubilized ACHE. The purified enzyme and peak I from starch-block electrophoresis also gave a single peak of activity (11--12 S) on centrifugation. The major molecular species present in white and grey matter appear to be very similar on the basis of their sedimentation coefficients irrespective of the method used to prepare the acetylcholinesterase. This is supported by the results obtained with gradient gel electrophoresis (Fig. 5) where the predominant form has a molecular weight of 260,000 which corresponds to a species of 12 S. These results agree with those of Hollunger and Niklasson (1973) and Viana et al., (1974) who respectively obtained molecular weights of 250,000 and 219,000 for the AChE from the caudate nucleus of bovine brain. Other forms detected by gradient gel electrophoresis were species with molecular weights of 180,000, 130,000 and 115,000 and these were present in both white and grey matter (Fig. 5). In addition to these forms there was also a species of MW 68,000 which was only present in the grey matter and could be a monomer of ACHE. Dudai and Silman (1972) found two types of monomer for the electric eel enzyme of molecular weights 59,000 and 85,000 and suggested that the smaller unit was an autolysis product of the larger one which in turn was associated into a tetramer of MW 320,000. The multiple molecular forms of AChE in pig brain can be considered to arise by aggregation of a monomer (MW 68,000) to form a dimer (MW 130,000) and a tetramer (MW 260,000) in agreement with previous findings (Mclntosh and Plummer, 1973; Chang and Blume, 1976). These could correspond to
the globular forms G,, G2 and G4 which seem to be present in a number of tissues, (Bon et al., 1979). The species of MW 180,000 is puzzling as it appears to be a trimer but this does not readily fit in with current ideas on the polymorphism of acetylcholinesterase (Massoulie, 1980; Massoulie et al., 1980). It may be a partially degraded molecule or be associated with other proteins although its presence in the purified preparation makes this unlikely. The species with a molecular weight of 115,000 may have arisen by proteolysis. The presence of such a range of molecular weight entities is at first surprising when compared with the results for sedimentation analysis, but such a range of oligomers could have arisen if an 'aggregating factor' (Kremzner and Fei, 1971) is removed during electrophoresis thus allowing the development of smaller molecular forms. The absence of species with a molecular weight greater than 260,000 in the purified preparations could likewise be due to the removal of such a factor during affinity chromatography. These results show that the multiple molecular forms of acetylcholinesterase are essentially the same in the white and the grey matter of porcine brain. The one exception to this is the species with a molecular weight of 68,000 which is present in the grey but not the white matter. The sole difference between the axonal and synaptic AChE in the white and grey matter of the pig brain therefore is the absence of the 68,000 MW species from the white matter. Acknowledyement--C. J. V. wishes to thank the Spanish Fundacion Cultural-Privada 'Esteban Romero' for their financial support.
REFERENCES Bon, S., Vigny, M. and Massoulie, J. 0979). Assymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. natn. Acad. Sei. U.S.A. 76, 2546--2550. Chan, S. L., Shirachi, D. Y., Bhargava, H. N., Gardner, G. and Trevor, A. J. 0972). Purifications and properties of multiple forms of brain acetylcholinesterase. J. Neurochem. 19, 2747--2758. Chandarajan, J. and Klein, L. (1975). Lowry assay of dilute proteins solutions containing high concentrations of Triton X-100. Analyt. Biochem. 69, 632-636. Chang, C. H. and Blume, A. J. (1976). Heterogeneity of acetylcholinesterasein neuroblastoma. J. Neurochem. 27, 1427-1435. Davis, G. A. and Agranoff, B. A. 0968). Metabolic behaviour of isozymes of acetylcholinesterase. Nature, Lond. 220, 277-280. Devonshire, A. J. (1975). Studies of the acetylcholinesterase from house flies (Musca domestica L) resistant and susceptible to organophosphorus insecticides. Biochem. J. 149, 463-469.
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whole-cell preparations of brain, muscle and liver. J. Dudai, Y. and Silman, I. (1971). The subunits of an acetylNeurochem. 5, 135-144. cholinesterase preparation purified from trypsin-treated Mclntosh, C. H. S. and Plummer, D. T. (1973). Multiple electric eel tissue. FEBS Lett. 16, 324-328. forms of acetylcholinesterase from pig brain. Biochem. J. Dudai, Y. and Silman, I. (1974). Acetylcholinesterase. Meth. 133, 655-665. Enzym. 34, 571-580. Ellman, G. L., Courtney, K. D., Andres, V. Jr. and Feather- Mclntosh, C. H. S. and Plummer, D. T. (1976). The subcellular localisation of acetylcholinesterase and its molstone, H. M. (1961). A new and rapid colorimetric deterecular forms in pig cerebral cortex. J. Neurochem. 27, mination of acetylcholinesterase activity. Biochem. Phar449-457. mac. 7, 88-95. Gordon, M. A., Chan, S. L. and Trevor, A. J. (1976). Active- Meunier, J. C., Olsen, R. W. and Changeux, J. P. (1972). Studies on the cholinergic receptor proteins from elecsite determinations on forms of mammalian brain and trophorus electricus. FEBS Lett. 24, 63-68. eel acetylcholinesterase. Biochem. J. 157, 69-76. Nachmansohn, D. (1971). Proteins in bioelectricity acetylHartree, E. F. (1972). Determination of proteins. A modificholinesterase and receptor. In: Handbook of Sensory cation of the Lowry method that gives a linear photoPhysiology Lowenstein, W. R. (Ed.) Vol. 1, pp. 18-102, metric response. Analyt. Biochem. 48, 422--427. Springer Verlag, New York. Helenius, A. and Simons, K. (1975). Solubilization of memOderfeld-Nowak, B. and Skangiel-Kramska, J. (1976). branes by detergents. Biochim. biophys. Acta 415, 29-79. Alterations in acetylcholinesterase isoenzyme pattern of Ho, I. K. and Ellman, G. L. (1969). Triton solubilized hippocampus after septal lesions in rat brain. J. Neuroacetylcholinesterase of brain. J. Neurochem. 16, chem. 27, 1241-1244. 1505-1513. Ott, P. and Brodbeck, U. (1978). Multiple molecular forms Hollunger, E. G. and Niklasson, B. H. (1973). The release of acetylcholinesterase from human erythrocyte memand molecular state of mammalian brain acetylcholinebranes. Eur. J. Biochem. 88, 11%125. sterase. J. Neurochem. 20, 821-836. Plummer, D. T. (1978). An introduction to Practical BioJackson, R. L. and Aprison, M. H. (1966). Mammalian chemistry, 2nd. Edn. pp. 284-287, McGraw-Hill, Lonbrain acetylcholinesterase Effects of surface active don. agents. J. Neurochem. 13, 1351-1365. Koelle, G. B. (1951). The elimination of enzymatic diffusion Plummer, D. T., Reavill, C. A. and Mcintosh, C. H. S. (1975). Properties of the soluble and membrane-bound artifacts in histochemical localisation of cholinesterases forms of acetylcholinesterase present in pig brain. Croaand a survey of their cellular distributions. J. Pharmac. tica chemica acta 47, 211-223. exp. Ther. 103, 153-171. Kremzner, L. T. and Fei, S. C. (1971). An acetylcholine- Reavill, C. A. and Plummer, D. T. (1978). Purification of acetylcholinesterase from pig cerebral cortex by affinity sterase aggregating protein. Fedn Proc. 30, 1193. chromatography. J. Chromat. 157, 141-151. Lewis, P. R. and Shute, C. C. D. (1966). The distribution of Rieger, F. and Vigny, M. (1976). Solubilization and physiocholinesterase in cholinergic neurons demonstrated with chemical characterisation of rat brain acetylcholinethe electron microscope. J. Cell Sci. 1, 381-390. sterase: development and maturation of its molecular Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin Phenol forms. J. Neurochem. 27, 121-129. Silver, A. (1974). The Biology of Cholinesterases. North reagent. J. biol. Chem. 193, 265-275. Maddy, A. H. and Dunn, M. J. (1973) The interpretation of Holland Publishing, Amsterdam. molecular weight data on membrane proteins. In: Pro- Spatz, L. and Strittmatter, P. (1971). A form of cytochrome tides of the Biological Fluids Peeters, H. (Ed.) Vol. 21, pp. b5 that contains an additional hydrophobic sequence of 21-26, Pergamon Press Oxford. 40 amino acid residues. Proc. natn. Acad. Sci. U.S.A. 68, 1042-1046. Margolis, J. and Kenrick, K. G. (1968). Polyacrylamide-gel electrophoresis in a continuous molecular sieve gradient. Snary, D., Goodfellow, P., Hayman, M. J., Bodmer, W. F. Analyt. Biochem. 25, 347-362. and Crumpton, M. J. (1974). Subcellular separation and Marples, E. A., Thompson, R. H. S. and Webster, G. R. molecular nature of human histocompatability antigens (1959). The liberation of active enzymes from brain tissue (HL-A). Nature 247, 457~,61. by lysolecithin. J. Neurochem. 4, 62-70. Srinivasan, R., Karczmar, A. and Bernsohn, J. (1972). ActiMartin, R. G. and Ames, B. N. (1961). A method for vation of acetylcholinesterase by Triton X-100. Biochim. determining the sedimentation behaviour of enzymes. biophys. Acta 284, 34%354. Applications to protein mixtures. J. biol. Chem. 236, Viana, S. B., Chan, S. L. and Trevor, A. J. (1974). Conver1372-1379. sions and stabilization of multiple forms of brain acetylcholinesterase. Proc. West. Pharmac. Soc. 17, 146-149. Massouli6, J. (1980). The polymorphism of cholinesterases and its physiological significance. TIBS 5, 160-164. Vijayan, V. J. and Brownson, R. H. (1974). Polyacrylamide gel electrophoresis of rat brain acetylcholinesterase: Massouli6, J., Bon, S. and Vigny, M. (1980). The polymorphism of cholinesterases in vertebrates. Neurochem. Intl isoenzymes of normal rat brain. J. Neurochem. 23, 2, 161-184. 47-53. McArdle, B., Thompson, R. H. S. and Webster, G. R. Wenthold, R. J., Mahler, H. R. and Moore, W. J. (1974). (1960). The action of lysolecithin and of snake venom on Properties of acetylcholinesterase from rat brain. J. Neurochem. 22, 945-949.
N.C.I. 3/5--D
0197-0186/81/050311-11 $02.00/0 © 1981 Pergamon Press Ltd.
MOLECULAR FORMS OF ACETYLCHOLINESTERASE PRESENT IN THE WHITE AND GREY MATTER OF PIG BRAIN MARGARETS. Y. CHAI, CHARLESA. REAVILL*,CECELIOJ. VIDAL~ and DAVID T. PLUMMER Department of Biochemistry, Chelsea College, University of London, Manresa Road, London SW3 6LX, U.K.
(Received 16 March 1981; accepted 13 July 1981) Abstract--Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove aeetyicholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter. The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter. Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge. Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed. A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.
Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7.) has been found in the central nervous system of all vertebrates studied, although the distribution of the enzyme in the brain is very uneven (Silver, 1979). The AChE in mammalian brain is known to exist in multiple molecular forms which have been identified by a variety of sedimentation, chromatographic and electrophoretic techniques. The multiple forms appear to differ in size, since gel filtration has revealed the presence of a number of species with a range of molecular weights (Chan et al., 1972; Wenthold et al., 1974; Gordon et al., 1976). This has been confirmed
by centrifugation studies which showed the enzyme to exist in forms with different sedimentation constants (Rieger and Vigny, 1976). The molecular forms of brain acetylcholinesterase also seem to differ in terms of their electrical charge as revealed by several electrophoretic techniques (Davis and Agranoff, 1968; Srinivason et al., 1972; Vijayan and Brownson, 1974). The existence of multiple molecular forms of acetylcholinesterase is thus well documented, but most workers have used the whole brain or the cortical region for their enzyme preparations, and there is little information on the properties of acetylcholinesterase and the molecular species present in different Present addresses: * Department of Neurology, Institute regions of the brain. For example, studies in our own laboratory have been carried out on pig cerebral corof Psychiatry Denmark Hill, London SE5, U.K. tex (Mclntosh and Plummer, 1973; Plummer et al., t Departmento Interfacultativo de Bioquinica, Facuitad de Medicina, Espinardo-Murcia, Spain. 1975) and subcellular fractions of this tissue (Mcln311
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MARGAREI S. Y. CHM et al.
tosh a n d P l u m m e t , 1976) but o t h e r regions of pig brain have n o t been examined. This work has n o w been e x t e n d e d to a study of A C h E in the white m a t t e r o f pig brain and the results are c o m p a r e d with the e n z y m e o b t a i n e d from the grey matter. T h e study o f acetylcholinesterase from the two parts of the brain should therefore p r o v i d e a crude but nevertheless useful c o m p a r i s o n o f axonal (white matter) a n d synaptic (grey matter) acetylcholinesterase.
EXPERIMENTAL Materials Porcine brains were obtained from the Co-operative Society Slaughter House, Woolwich, London. Potato starch was supplied by BDH, Poole, Dorset and fine chemicals were purchased from Sigma, London. The acetylcholinesterase inhibitor BW 284 c51 was obtained from Burroughs Wellcome, Beckenham, Kent. Ethopropazinc was supplied as a gift from May & Baker Ltd. All solutions were prepared in distilled water, redistilled from an all glass still. Assay of acetylcholinesterase The activity of acetylcholinesterase was determined at 30°C and pH 7.9 using a pH-stat (Mclntosh and Plummer, 1976). A colorimetric method was employed when a large number of fractions had to be assayed as in starch-block electrophoresis and density gradient centrifugation. In this case, the substrate used was acetylthiocholine and the mercaptide formed from the hydrolysed thioester was reacted with an oxidizing agent 5,5'dithio-(bis)-(2-nitrobenzoic acid), DTNB to give a product that absorbs at 412 nm (Ellman et al., 1961). Protein assay The protein content of a membrane suspension was measured by a modified biuret method where 1.5~o w/v sodium deoxycholate was incorporated into the reagent to solubilize all of the proteins present. The biuret reagent (2ml) was added to the protein solution (1 ml) and the mixture heated on a boiling water bath for 1 rain. At the end of this period, the mixture was cooled to room temperature and the extinction measured at 540 nm. The method of Lowry et al. (1951) was used when low concentrations of protein were present. The detergent Triton X-100 interferes with this assay, but this was overcome by centrifuging the precipitate (10000, 5min) and incorporating the detergent in the reagent blank and standards (Hartree, 1972; Chandarajan and Klein, 1975). Solubilization of the enzyme All solubilization procedures were performed on porcine brains obtained on the day of use from the slaughter house. The membranes and blood vessels were removed from the surface of the brains and the white and grey matter separated and dispersed in a Waring Blender for 5 rain at 4°C with 30 mM sodium phosphate buffer (pH 7.0) to produce a 10~o homogenate (g wet weight per ml). Centrifugations were then carried out on an MSE SS 50 preparative ultracentrifuge using an 8 × 50ml or a 10 × 10ml capacity
rotor. The enzyme was considered to be soluble if it remained in the supernatant after being centrifuged al 100,000 o for 1 h. The supernatant from this extraction is known as the 'naturally soluble' form of the enzymc~ Further solubilization of acetylcholinesterase was carried out by resuspending the pellet in an appropriate medium and gently stirring. The extract was then centrifuged at 100,000 0 for 1 h and the supernatant assayed for AChE activity. Triton X-100, lysolecithin and EDTA were prepared in 30raM sodium phosphate, pH 7 while sodium cholate and sodium deoxycholate were dissolved in 50 mM Tris buffer pH 7.5. The condilions varied slightly according to the solubilizing agent employed and 30 min at 37 C was found to be optimal for the naturally occurring detergents, sodium cholate, sodium deoxycholate and lysolecithin (Marples et al., 1959; McArdle et al., 1960). The synthetic detergent Triton X-100 (Ho and Ellman, 1969) needed only 10rain at room temperature, while EDTA extraction (Chan et al., 1972; Hollunger and Niklasson, 1973) required stirring for 2 h at 4 C . In the latter case, the supernatant was removed and the pellet treated twice more in the same way. The concentration of the reagents used are given in Fig. 1. A~hnity ehromatooraphy The AChE was purified on an affinity column of Sepharose 4B linked to the ligand (1-methyl-9-(Na-~-aminohexanoyl)-fl-aminopropylamino)-acridinium bromide hydrobromide (Dudai and Silman, 1974). The preparation of this column (MAC-agarose) and the method used was the same as that previously described for the purification of AChE from pig cerebral cortex (Reavill and Plummer, 1978). Detergent was present throughout the preparation and the purified enzyme contained 0.1°/,, w/v Triton X-100. Starch-block electrophoresis Several of the enzyme preparations were subjected to electrophoresis on starch grains (Plummer, 1978). Potato starch was washed four times by decantation; twice with water and twice with the electrophoresis buffer (sodium phosphate, 0.1 M, pH 7). In some experiments, Triton X-100 was incorporated into the buffer at a final concentration of 1°/,~ w/v. Excess buffer was decanted and the starch pressed into a block with the aid of a Perspex former. Wicks consisting of several layers of muslin were used to connect the block with the buffer in the electrode tanks (0.1 M, sodium phosphate pH 7). The power pack was then connected and the block equilibrated at 6 V cm-for I h at 4°C. The enzyme sample containing 5-10 mg of protein was then applied to a laterally cut groove, 0.5 cm wide towards the cathode end of the block and bovine serum albumin stained with bromphenol blue was applied as a marker in a cavity in line with the groove. The cavity and groove were filled with starch and electrophoresis carried out at 6 - 7 V c m -~ for 18h. The position of the markers was carefully noted and the longitudinal section containing them discarded. The remainder of the block was cut transversely into strips 0.5 or 1 cm wide, placed in a sintered glass funnel and the enzyme elute~l with 2 ml aliquots of buffer. The acetylcholinesterase in each fraction was then assayed by the EIlman method.
Acetylcholinesterase in pig brain
Polyacrylamide-oel-electrophoresis Multiple forms of acetylcholinesterase were separated on the basis of differences in size by prolonged electrophoresis on a concave gradient of polyacrylamide (Margolis and Kenrick, 1968; McIntosh and Plummer, 1973). The gels were then stained for enzyme activity with acetylthiocholine as substrate by the method of Koelle (1951) as modified by Lewis and Shute (1966).
Density gradient centrifugation Enzyme samples were placed on top of a linear sucrose gradient (5 to 60~o w/v) prepared in 5 ml polycarbonate tubes then centrifuged overnight (17h) at 100,000g. The standard protein bovine catalase with a sedimentation coefficient of 11.4 S and a molecular weight of 240,000 was mixed with the enzyme sample to act as a convenient marker. At the end of the centrifugation, the tubes were punctured with an M.S.E. tube piercer and fractions collected and assayed for acetylcholinesterase activity. Catalase was assayed by following the decrease in extinction at 240 nm of a mixture containing 3 ml sodium phosphate buffer (10 mM, pH 7.5), 20 #1 of hydrogen peroxide (0.9 M) and 20/tl of the fraction. Activities were calculated in terms of the change in absorbance per minute and the fraction with maximum catalase activity was noted. The efficacy of the technique was tested by running standard proteins on the gradients and checking that a linear response was obtained when the sedimentation constants were plotted against the distance migrated down the tube (Martin and Ames, 1961). The sedimentation constant of each peak of enzyme activity was calculated from the distance travelled down the tube relative to the catalase marker (Martin and Ames, 1961). A crude estimate of the molecular weight of each species was obtained from the formula: $1/$2 = (MWI/MW2) 2/3 when $1 = sedimentation constant of unknown, $2 = sedimentation constant of catalase (11.4S), MW1 = molecular weight of unknown and MW2 = molecular weight of catalase (240,000). RESULTS
Solubilization of acetylcholinesterase from white matter Acetylcholinesterase is a membrane-bound enzyme (Nachmansohn, 1971) and so the initial experiments involved the extraction of the white matter with a variety of agents in an attempt to bring the enzyme into solution. The AChE activity in the uncentrifuged homogenate is taken to be 100~ and the enzyme was considered to be soluble if it remained in the supernatant after centrifugation at 100,000 g for 1 h. Dilute buffer solution. Extraction of white matter with water or dilute buffer (30 mM sodium phosphate, pH 7.0) solubilized from 13 to 179/o of the total activity. This fraction was referred to as the 'naturally soluble' enzyme and subsequent extraction of the pellet did not bring any more enzyme into solution. Attempts were then made to solubilize the membrane-bound enzyme by extracting the pellet with a variety of media.
313
EDTA. Repeated homogenization and incubation with 3 0 m M sodium phosphate buffer containing 1 m M EDTA brought 39/0 of the enzyme into solution (range 2.8-3.29/0). The yield was not improved by altering the concentration of the brain homogenate. Triton X-IO0. The non-ionic detergent Triton X-100 was not very effective as a solubilizing agent and as the total Triton X-100 concentration was raised from 0.1 to 2~o w/v, the amount of AChE solubilized increased from 16 to only 18~o of the total homogenate activity. Lysolecithin. Lysolecithin was a more effective solubilizing agent than EDTA or Triton X-100 and when the 100,000 g pellet was extracted with 12 m M lysolecithin, about 33~o (range 30-36~o) of the total homogenate activity was solubilized from the pellet. Bile salts. Bile salts were then used in an attempt to bring the enzyme into solution and the early results with sodium cholate were disappointing. Extraction of the 100,000g pellet with 1~o w/v sodium cholate solubilized only 18.5~o (range 16.5-20.59/o) of the AChE activity. However, sodium deoxycholate was very effective as a solubilizing agent and a 0.2~o w/v concentration of the detergent brought 699/0 (range 66-72~o) of the total homogenate activity into solution. Increasing the concentration of sodium deoxycholate, solubilized more of the enzyme with a maximum yield of 88~o (range 87-89~o) using 0.3~ w/v sodium deoxycholate. Therefore all of the AChE activity of the homogenate of white matter could be accounted for with 15~o extracted with dilute buffer and 88~ with 0.3~o w/v sodium deoxycholate. However, although concentrations of detergent greater than 0.2~o w/v solubilized more of the enzyme, the problem of lipid coming out of solution made it impracticable for routine use so that extraction with 0.2~o w/v sodium deoxycholate was adopted as the standard procedure for white matter. There was essentially no difference in the AChE activity between a 10~o w/v suspension of the white matter of porcine brain in dilute buffer and a 10~o w/v suspension of brain in any of the solubilizing agents used, indicating the absence of activation or inhibition. A summary of these results and a comparison with the grey matter of brain is shown in Fig. 1 and Table 1. Specificity of the enzyme preparations The substrate specificities of the sodium deoxycholate solubilized acetylcholinesterase and the naturally soluble enzyme were examined by measuring the rates of hydrolysis of a number of substrates and compar-
314
MARGARE]
%
0 A c t i v i t y in IO0,OOg
al.
S. Y. C H A I e t
Total octiv,ty solubilized from the IO0, O00g pellet 20
40
60
80
pellet
IC,C~ I
E xtroction method
I
EDTA (traM}
]
Triton X - I00 (1% w/v)
]
Lysotecithin (12 mM) Sodium eholote (1% w/v) Sodium deoxycholote (0.2 % w/v)
Fig. 1. The solubilization of membrane-bound acetylcholinesterase from the white (11) and grey matter (E]) of porcine brain. The total enzyme activity in the homogenate is shown as 100~o which is made up of 15°/o that can be extracted with dilute buffer and 85,% which is membrane-bound ( . . . . . . . ). The amount of enzyme solubilized is that which remains in the supernatant after centrifugation at 100,000 g for 1 h. Details of the extraction procedures are given in the text. ing them with acetylcholine. The results obtained show that the 'naturally soluble' and the detergent solubilized preparations had very similar specificities (Table 2). Data obtained for the acetylcholinesterase in porcine grey matter are also included for comparison. The Michaelis constants (K,,) were measured for the crude homogenate, the naturally soluble and the detergent solubilized forms of the enzyme from pig brain and the results are shown in Table 1. In the case of the white matter, the naturally soluble form of the enzyme had the highest K,, with the homogenate significantly lower than this and the detergent solubilized enzyme slightly less than the homogenate value.
The Michaelis constants for the grey matter were the same as those for the corresponding preparations from the white matter of porcine brain.
Purification by affinity chromatography The deoxycholate solubilized preparation (100150 ml) was applied to the affinity column at a rate of about 3 0 m l h -1. Following this, the column was washed with the elution buffer (30 raM, sodium phosphate, pH (7.0) until no protein could be detected in the eluate, The acetylcholinesterase inhibitor, decamethonium bromide ( l O m M ) w a s then incorporated into the buffer and the column washed until no further enzyme could be detected in the fractions. The
Table 1. Enzyme activity and Michaelis constants of AChE prepared from the white and grey matter of pig brain
Preparation
Acetylcholinesterase activity (•moles min- 1) per g wet weight (o(,) per mg protein
Michaelis constants (pM), acetylcholine
Homogenate White matter Grey matter
3.80 5.11
(100) (100)
0.048 0.082
70 76
Naturally soluble enzyme White matter Grey matter
0.58 0.71
(15) (14)
0.030 0.028
106 118
Detergent solubilized enzyme White matter Grey matter
2.60 2.85
(69) (56)
0.062 0.142
64 62
The values shown are the mean of 4-6 experiments.
315
Acetylcholinesterase in pig brain 0.6
I
NaCI
Decamethoniurn bromide (10 mM) /
Z.
E E
0.~
_=
E rr
=L
E
0.2
E
c~
t3
E LU 120
240
Elution volume,
5bO
rnL
Fig. 2. Elution profile of acetylcholinesterase from a column of MAC-agarose. The elution medium used was 30 mM sodium phosphate buffer, pH 7.0 and the arrows mark the point at which the elution medium was changed to include the reagents shown. Experimental details are as in the text. eluting medium was then changed to sodium phosphate buffer containing 1 M NaCI when a further peak of enzyme activity was obtained. The fractions containing the enzyme were dialysed (Reavill and Piummer, 1978) and the activity deter-
mined. The elution profile is shown in Fig. 2 and the AChE activity of the peaks in Table 3. The total yield of the enzyme from the column was 35~o with 24~o of the enzyme activity eluted in the decamethonium fraction (peak I) and 11~o of the activity eluted by 1 M
Table 2. The relative hydrolysis of choline esters by brain acetylcholinesterase
Buffer soluble enzyme
Substrate Acetylcholine iodide
Activity as % of acetylcholine iodide White matter Grey matter Detergent soluble Detergent soluble enzyme Buffer enzyme (0.2% w/v sodium) soluble (1% w/v (deoxycholate) enzyme Triton X-100)
100
Propionylcholine iodide Butyrylcholine iodide Acetyl=fl-methylcholine bromide Tributyrin
100
100
82.4
80.9
2.0
100
83.0
84.0
1.8
0
0
21.3
22.0
20.0
22.0
7.4
8.0
4.0
5.0
The substrate concentration in all cases was 1 mM and the activity was determined with a pH-stat as described in the text. The rate of hydrolysis is expressed as a percentage of that found with acetylcholine iodide. The values shown are the average of 4 experiments. Table 3. The purification of acetylcholinesterase from the white matter of pig brain on an affinity column of MAC-agarose
Total applied
Protein (mg)
Enzyme activity (#)
Specific activity (# mg- t protein)
Yield (%)
Purification (times)
629.0
39.84
0.063
--
--
Recovered in peak I
0.52
9.69
18.63
24.0
300
Recovered in peak II
0.36
4.55
12.64
11.0
200
The units of enzyme activity (#) represent ~moles acetylthiocholine iodide hydrolysed per minute.
316
M A R G A R E T S. Y . C H A I e l
020 JA2
(a} Deoxycholote - solubilized enzyme
L E Ol5 'c E ::::L
010
>~ > 005
~ >.
0
I
1
ezm;
(b) Naturelly soluble
0 05[
0
I
5
~o
Distance from anode,
115
I 2o
cm
Fig. 3. Starch-block electrophoresis of acetylcholinesterase from the white matter of porcine brain. (a) Deoxycholate solubilized enzyme. (b) Naturally soluble enzyme. The arrow .L denotes the point of application of the sample while Alb marks the position of the albumin marker. Experimental details as in the text. NaC1 in the second peak (II). The purification of the acetylcholinesterase in peak I was 300 fold while that in Peak II was 200 fold. The enzyme in the first peak was therefore used in subsequent investigations.
Electrophoresis Starch-block electrophoresis. The naturally soluble acetylcholinesterase from the white matter showed one peak of enzyme activity and the deoxycholate solubilized enzyme also gave only one peak of enzyme activity with a similar electrophoretic mobility to that of the soluble enzyme (Fig. 3). In contrast to this, the Triton solubilized enzyme from grey matter gave a very broad band of activity with a low electrophoretic mobility (Fig. 4a). However, if 1% w/v Triton X-100 was incorporated into the block and buffer system, then two peaks of activity were obtained, which was the same as the pattern obtained for the naturally soluble preparation from grey matter (Fig. 4b). The fast moving peaks from all the preparations showed a range of molecular weights during electrophoresis on a gradient of polyacrylamide, whereas the slow moving peaks present in the grey matter appeared to be
~ll.
an aggregated form of the enzyme with a high molecular weight. This is because peak 11 from the starchblock barely entered the gradient gels giving an intense surface staining and also migrated to the bottom of the centrifuge tube during density gradient centrifugation. The purified enzymes showed the same characteristics as the detergent solubilized enzymes when subjected to electrophoresis on a starch-block. Gradient polyacrylamide-gel electrophoresis. Electrophoresis on a gradient of polyacrylamide showed multiple bands of enzyme activity in all the enzyme preparations examined. The results are shown in Fig. 5 where the approximate molecular weights are indicated on the assumption that the acetylcholinesterase molecules are spherical. The naturally soluble enzyme from the white matter had two major bands of activity with molecular weights of 660,000 and 260,000 a less intense but broad band with a molecular weight of 120,000 and a faintly staining band at 400,000. The sodium deoxycholate solubilized enzyme had three bands of activity in common with the naturally soluble acetylcholinesterase (400,000, 260,000, 120,000) and also a species
AIb
]I
-EL(O) Triton - solubilized enzyme
004 L E
[[
E
0 02
r \
o E
>
c
0
04
(b) Naturally soluble
enzyme
002
0
F
l/<
I0
Distonce from onode,
20
30
cm
Fig. 4. Starch-block electrophoresis of acetylcholinesterase from the grey matter of porcine brain. (a) Triton solubilized enzyme. (b) Naturally soluble enzyme. Other details as in Fig. 3.
Acetylcholinesterase in pig brain Detergent solubilized
Noturolly soluble w
W
G
G
317
Purified enzyme W
G
Molecular weiqhts II
I
I
I I
-
-[] i
• I
- -
i
[]
- -
--
-
I
-
i
-
660,000
400, 000 z6o, ooo 180, o o o 130, 000
68,000
I
m
Fig. 5. Electrophoresis of acetylcholinesterase preparations from porcine brain on a gradient of polyacrylamide. W denotes white matter and G the grey matter. Depth of staining: strong I ; moderate l ; light m. Experimental details as in the text. with a MW of 180,000. The peaks of activity from the starch-block electrophoresis of both enzyme preparations gave the same staining patterns as those shown in Fig. 5. The staining pattern for the purified enzymes was very similar to the deoxycholate extract, but the bands were sharper and more intensely stained than in the other preparations. There were, however, some minor differences in that the faint band at 400,000 disappeared on purification while the broad band with an average molecular weight of 120,000 was resolved into two species with molecular weights of 115,000 and 130,000. The results for the grey matter are also shown in Fig. 5 and the differences and similarities to the patterns obtained for the white matter preparations will be considered in the discussion. When 10pM eserine was incorporated into the staining system, no AChE activity was detected showing that the bands of activity were due to cholinesterase. If the acetylthiocholine iodide was substituted by butyrylthiocholine iodide, a few faint bands of activity were seen, but none of them corresponded to the bands shown in Fig. 5. The activity detected therefore appears to be due to acetylcholinesterase rather than butyrlcholinesterase. Finally, the incorporation of 30 pM ethopropazine into the staining system had no effect on the bands produced when acetylthiocholine was used as the substrate confirming that the enzyme detected was acetylcholinesterase.
Density gradient centrifugation White matter. The naturally soluble form of the
enzyme gave one peak of activity with a sedimentation constant of 9.5 S (Fig. 6b). The results for the deoxycholate solubilized enzyme was very similar with one peak at 10.5 S provided that sodium deoxycholate was incorporated into the sucrose (Fig. 6a). If the detergent was omitted, then the AChE sedimented to the bottom of the tube indicating the presence of a high molecular weight species. In view of this, detergent was always present during centrifugation of the deoxycholate-solubilized enzyme. The peaks of activity obtained from the starchblock electrophoresis of both enzyme preparations also gave only one peak of activity with a sedimentation coefficient of about 11 S. The purified enzyme when centrifuged on a sucrose gradient was found to have the same sedimentation constant as the crude enzyme solubilized with 0.2~o w/v sodium deoxycholate. Grey matter. When fresh preparations of Triton solubilized AChE were centrifuged on sucrose gradients, one major peak of activity was obtained corresponding to 11-12S. The naturally soluble acetylcholinesterase consistently showed a major peak of activity at 11-12 S, and there was also a suggestion of some minor shoulders indicating the presence of higher molecular weight species, but the resolution was insufficient to enable sedimentation constants to be assigned to them with any degree of accuracy. The peaks of the acetylcholinesterase preparations obtained by starch-block electrophoresis were also subjected to density gradient centrifugation. In all cases, peak II migrated rapidly to the bottom of the gradient indicating a high molecular weight species
318
MARGARI-TS. Y. CHAIet al. Catalase 1145 0to-
(a} Deoxycholate-solubilized
enzyme
E g c o
g o c +-
005
•
/
2 to
./
,
~
.e/
,, e . , ~
c
o= o >"
~ Catolase 11.45
005
t~
"~
0
q
(b) Naturally soluble
enzyme
2 3 4 Fraction, mt
5
Fig. 6. Sucrose density gradient centrifugation of acetylcholinesterase prepared from the white matter of porcine brain. I denotes the position of the catalase marker (l 1.4 S). The top of the tube is on the left of the diagram so the sedimentation direction is as shown (--*). Experimental details as in the text. greater than 1 x 106. Peak 1 on the other hand showed a major band of activity at 11-12 S. Acetylcholinesterase purified by affinity chromatography was also centrifuged on a sucrose gradient and was found to have the same sedimentation characteristics as the crude Triton solubilized enzyme both before and after starch-block electrophoresis. DISCUSSION Acetylcholinesterase is present in the white matter of porcine brain in significant quantities, although the activity per g wet weight or per mg protein is less than that found in the grey matter (Table 1). Extraction of the white matter of pig brain with dilute buffer solution or water solubilized 15~ of the total acetyicholinesterase activity present in the tissue. This value for the 'naturally soluble' form of the enzyme is very similar to that obtained by other
workers using other sources of the enzyme (Ho and Ellman, 1969; Devonshire, 1975; Oderfeld-Nowak and Skangiel-Kramska, t976: McIntosh and Plummer, 1973). The soluble form of the enzyme does not appear to be in equilibrium with the membrane enzyme, since no more enzyme was brought into solution when the 100,000 0' pellet was re-extracted with buffer. Identical results were obtained for the grey matter so the relative amount of soluble AChE and its specific activity appears to be the same in both regions of the brain (Table 1) although its precise function is not known. Several attempts were then made to bring the membrane-bound form of the enzyme into solution by extracting the 100,000 0 pellet with a variety of agents. Ethylenediaminetetraacetic acid (EDTAt is known to facilitate the removal of proteins from membranes probably by disrupting the divalent ion bridges which stabilize protein-lipid complexes (Hollunger and Niklasson, 1973; Maddy and Dunn, 1973). Several groups have, therefore, used repeated extraction with EDTA to solubilize AChE and obtained up to 70}o of the enzyme in a soluble form (Chan et al., 1972; Hollunger and Niklasson, 1973: Wenthold et al., 1974) but in the case of the white matter, only 3% could be solubilized with EDTA. The association between AChE and the membrane seems to be quite strong and so detergents were used as these are usually successful at solubilizing proteins, which are firmly bound to the lipid matrix of membranes (Helenius and Simons, 1975). Extraction with the non-ionic detergent Triton X-100 was not very effective, since only 17"~i of the enzyme activity could be extracted with a concentration of 1~,,;w/v. This is in marked contrast to the 85}/o solubilization obtained for AChE from rat brain (Ho and Ellman, 1969). Lysolecithin was better than Triton X-100 as a solubilizing agent and extraction with a 12 mM concentration of the detergent resulted in the release of 33}0 of the total homogenate activity from the pellet. The yield, however, was still rather low, so for this reason and the expense, lysolecithin was not used as a routine solubilizing agent. Bile salts have been successfully used to solubilize many membrane proteins without loss of biological activity (Spatz and Strittmatter, 1971; Meunier et al., 1972; Snary et al, 1974) and since they are stronger detergents than Triton X-100 or lysolecithin it was felt that cholate or deoxycholate might give a good yield of soluble ACHE. Extraction with 1~ w/v sodium cholate was disappointing, with a yield of 19~o of the enzyme activity, virtually the same as that obtained with Triton X-100. However, the results with sodium
Acetylcholinesterase in pig brain deoxycholate were very satisfactory and a 0.3~o w/v solution of the bile salt solubilized all of the AChE activity in the pellet. Unfortunately, lipid material came out of solution at this concentration which made it difficult to remove the supernatant and so a concentration of 0.2 w/v deoxycholate was routinely used which avoided this problem. The results summarized in Fig. 1 show that when the tissue was extracted with EDTA, lysolecithin or Triton X-100, much lower yields were obtained for the white matter than the grey. Furthermore, the strong detergent sodium deoxycholate was needed to solubilize the AChE in white matter whereas the mild detergent Triton X-100 which was satisfactory for the grey matter was unable to dissociate the enzyme from the membrane in sufficient yield. These results suggest that the AChE in the white matter of porcine brain is more firmly bound to the membranes than the enzyme in the grey matter. Acetylcholinesterase was purified by affinity chromatography on a column of MAC-agarose that was originally developed by Dudai and Silman (1974) for the purification of AChE ~'rom electric eel. The AChE was firmly bound to the column and could be eluted with 10 mM decamethonium bromide (Fig. 2). The yield from this peak (I) was 24~o and the specific activity was 18.63 gmoles min- 1 mg protein- 1, a purification of about 300 fold (Table 3). When the Triton solubilized enzyme from grey matter was purified on the MAC-agarose column, the yield of enzyme in the decamethonium peak was 44yo with a specific activity of 147.6, a 900 fold purification (Reavill and Plummer, 1978). A second peak of enzyme activity (II) was eluted from the column with M NaC1 and accounted for a further 11~o of the enzyme activity. The grey matter also showed a second peak following elution with M NaC1 amounting to 13~o of the enzyme activity (Reavill and Plummer, 1978). In both cases, some binding to the column would seem to occur by electrostatic rather than biospecific interactions indicating a similarity in the two preparations. However, there are significant differences between the binding of the AChE from the white and grey matter to the column which could reflect the differences in the binding of the enzyme to membranes in vivo. The binding characteristics of the AChE from the white and grey matter may be different, but the kinetic properties of the enzyme from the two sources appear to be the same. The Michaelis constants (K,) for a particular preparation are in the same range (Table 1) and are similar to the values obtained from other species (80-140#M). (Jackson and Aprison, 1966; Ho and Ellman, 1969; Chan et al., 1972). The
319
specificity of the preparations from white and grey matter are also very similar and are typical of acetylcholinesterase (Table 2). Starch-block electrophoresis was used as a semipreparative separation of molecular forms which differ in charge. The deoxycholate solubilized AChE and the naturally soluble enzyme both gave a single peak on electrophoresis. However, the naturally soluble enzyme appears to have a slightly higher negative mobility than the bile salt solubilized enzyme (Fig. 3). The purified enzyme showed the same characteristics as the deoxycholate extract. In the case of the grey matter, two peaks of activity were obtained for the naturally soluble form of the enzyme with a fast migrating peak (I) as the major species (Fig. 4b). The same pattern was obtained for the membrane enzyme after it had been solubilized with Triton X-100 provided that detergent was present in the electrophoresis block and buffer. However, if the detergent was excluded from the block then a zone of activity was obtained corresponding to peak II (Fig. 4a). In all cases, peak II sedimented to the bottom of the tube during density gradient centrifugation and barely penetrated the polyacrylamide gel during electrophoresis (Fig. 5). This suggests that the molecular weight of the AChE in peak II is very high and is probably an aggregate of acetylcholinesterase molecules. In contrast to this, sedimentation and electrophoretic measurements clearly showed that peak I was not an aggregated form of the enzyme. These results show that both the naturally soluble and the Triton solubilized forms of the enzyme from grey matter have a tendency to aggregate and that this is most marked in the case of the membrane enzyme soluzilized with Triton. In the absence of Triton X-100, virtually complete aggregation occurs (Fig. 4a) so that detergent is necessary to keep the enzyme extracted from the membrane in solution. The hydrophic nature of some of the molecules of acetylcholinesterase has also been seen in other preparations (Ho and Ellman, 1969; Ott and Brodbeck, 1978) and discussed in recent reviews (Massoulie, 1980; Massoulie et al., 1980). In this present study the aggregated acetylcholinesterase (peak II) would not enter polyacrylamide gel even in the presence of lYo w/v Triton X-100 so it appears that the aggregation could not be reversed by detergent and may well be irreversible as suggested by Hollunger and Niklasson (1973). All four preparations of AChE showed only one unaggregated form of the enzyme (Figs. 3 and 4) with slight difference in the electrophoretic mobility for the enzyme from the white and the grey matter. The
320
MARGARETS. Y. (THAIet al.
single peak on electrophoresis shows the presence of only one charged entity and suggests that the molecular species of brain AChE do not arise from difference in charge. Centrifugation on a sucrose gradient was therefore used to see if AChE exists in multiple forms of different molecular weights. The naturally soluble AChE from white matter gave a single peak with sedimentation constant of 9.5 S whereas the deoxycholate solubilized enzyme migrated to the bottom of the tube indicating the presence of a highly aggregated species. However, if 0.2% w/v sodium deoxycholate was incorporated into the sucrose gradient, the aggregation was abolished and the enzyme resolved into a single peak of activity with a sedimentation constant of 10.5 S (Fig. 6). An identical result was obtained for the purified enzyme both before and after starch-block electrophoresis. Very similar results were obtained from the preparations from grey matter with a peak of 11 12 S. As with the white matter, detergent had to be present in the sucrose gradient to prevent aggregation of the Triton solubilized ACHE. The purified enzyme and peak I from starch-block electrophoresis also gave a single peak of activity (11--12 S) on centrifugation. The major molecular species present in white and grey matter appear to be very similar on the basis of their sedimentation coefficients irrespective of the method used to prepare the acetylcholinesterase. This is supported by the results obtained with gradient gel electrophoresis (Fig. 5) where the predominant form has a molecular weight of 260,000 which corresponds to a species of 12 S. These results agree with those of Hollunger and Niklasson (1973) and Viana et al., (1974) who respectively obtained molecular weights of 250,000 and 219,000 for the AChE from the caudate nucleus of bovine brain. Other forms detected by gradient gel electrophoresis were species with molecular weights of 180,000, 130,000 and 115,000 and these were present in both white and grey matter (Fig. 5). In addition to these forms there was also a species of MW 68,000 which was only present in the grey matter and could be a monomer of ACHE. Dudai and Silman (1972) found two types of monomer for the electric eel enzyme of molecular weights 59,000 and 85,000 and suggested that the smaller unit was an autolysis product of the larger one which in turn was associated into a tetramer of MW 320,000. The multiple molecular forms of AChE in pig brain can be considered to arise by aggregation of a monomer (MW 68,000) to form a dimer (MW 130,000) and a tetramer (MW 260,000) in agreement with previous findings (Mclntosh and Plummer, 1973; Chang and Blume, 1976). These could correspond to
the globular forms G,, G2 and G4 which seem to be present in a number of tissues, (Bon et al., 1979). The species of MW 180,000 is puzzling as it appears to be a trimer but this does not readily fit in with current ideas on the polymorphism of acetylcholinesterase (Massoulie, 1980; Massoulie et al., 1980). It may be a partially degraded molecule or be associated with other proteins although its presence in the purified preparation makes this unlikely. The species with a molecular weight of 115,000 may have arisen by proteolysis. The presence of such a range of molecular weight entities is at first surprising when compared with the results for sedimentation analysis, but such a range of oligomers could have arisen if an 'aggregating factor' (Kremzner and Fei, 1971) is removed during electrophoresis thus allowing the development of smaller molecular forms. The absence of species with a molecular weight greater than 260,000 in the purified preparations could likewise be due to the removal of such a factor during affinity chromatography. These results show that the multiple molecular forms of acetylcholinesterase are essentially the same in the white and the grey matter of porcine brain. The one exception to this is the species with a molecular weight of 68,000 which is present in the grey but not the white matter. The sole difference between the axonal and synaptic AChE in the white and grey matter of the pig brain therefore is the absence of the 68,000 MW species from the white matter. Acknowledyement--C. J. V. wishes to thank the Spanish Fundacion Cultural-Privada 'Esteban Romero' for their financial support.
REFERENCES Bon, S., Vigny, M. and Massoulie, J. 0979). Assymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. natn. Acad. Sei. U.S.A. 76, 2546--2550. Chan, S. L., Shirachi, D. Y., Bhargava, H. N., Gardner, G. and Trevor, A. J. 0972). Purifications and properties of multiple forms of brain acetylcholinesterase. J. Neurochem. 19, 2747--2758. Chandarajan, J. and Klein, L. (1975). Lowry assay of dilute proteins solutions containing high concentrations of Triton X-100. Analyt. Biochem. 69, 632-636. Chang, C. H. and Blume, A. J. (1976). Heterogeneity of acetylcholinesterasein neuroblastoma. J. Neurochem. 27, 1427-1435. Davis, G. A. and Agranoff, B. A. 0968). Metabolic behaviour of isozymes of acetylcholinesterase. Nature, Lond. 220, 277-280. Devonshire, A. J. (1975). Studies of the acetylcholinesterase from house flies (Musca domestica L) resistant and susceptible to organophosphorus insecticides. Biochem. J. 149, 463-469.
Acetylcholinesterase in pig brain
321
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