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Multiple Forms Of Soluble Butyrylcholinesterase In Human Brain
BIOCHIMICA ET BIOPHYSICA ACTA
477
BBA 35576
MULTIPLE FORMS OF SOLUBLE BUTYRYLCHOLINESTERASE IN HUMAN BRAIN
JENS B. CARLSEN* AND OLE SVENSIVIARK* The Institute of Neurophysiology, University of Copenhagen, Copenhagen (Denmark) (Received December 2gth, rg6g)
SUMMARY r. 30-55% of the butyrylcholinesterase (EC 3.r.r.8) activity of human brain was present in an aqueous extract. Particle-bound butyrylcholinesterase could not be made soluble. 2. In DEAE-cellulose chromatography soluble butyrylcholinesterase was separated in one inhomogeneous main fraction and several minor fractions. Gel chromatography on Sephadex G-200 resolved the main fraction into two inhomogeneous fractions, one of higher molecular weight (300 000-400 ooo), the other of lower molecular weight (so ooo-rso ooo). 3· Electrofocusing of the butyrylcholinesterase fraction of higher molecular weight revealed two inhomogeneous fractions in the pH ranges 3.8-4.7 and 5.6-8.2. By treatment with neuraminidase (EC 3.2.r. r8) the most acid subfractions disappeared and alkaline subfractions appeared. The acid and the most alkaline subfractions may originate from plasma trapped in the tissue. Electrofocusing of the fraction of lower molecular weight showed at least 6 subfractions in the pH range 5.6-8.0. Treatment with neuraminidase had no effect on their isoelectric points. 4· Brain tissue thus contains at least 4-5 soluble butyrylcholinesterase fractions of high molecular weight with isoelectric points ranging from 5.6 to J.O and at least 6 low-molecular-weight fractions with isoelectric points from s.6 to 8.0.
INTRODUCTION In electrophoresis on paperl, agar gel2, and starch geP· 4 soluble butyrylcholinesterase activity from human brain occurred in 2-3 bands with different mobilities. Preliminary experiments (I. M. PETERSEN, unpublished) have shown that human brain butyrylcholinesterase could be separated in at least 3 fractions by DEAE* Present address: Department of Biochemistry C, University of Copenhagen, 71 Radmandsgade, DK 2200 Copenhagen N, Denmark.
Biochim. Biophys. Acta, 207 (r970) 477-484
J. B. CARLSEN, 0. SVENSMARK
cellulose chromatography and gel filtration. In the study presented in this report an attempt was made to isolate these fractions of butyrylcholinesterase in amounts allowing investigation of their properties, especially with respect to their content of sialic acid, and to compare them with human serum butyrylcholinesterase. Furthermore, an attempt was made to solubilize the particle-bound butyrylcholinesterase activity of human brain. MATERIALS AND METHODS Brain tissue IS human brains were obtained I-3 days after death. The causes of death were accidents (8), coronary occlusion (5), cancer colli uteri (I), and renal failure (I). The whole brain except for the cerebellum was used. After the removal of membranes and large vessels, the tissue was rinsed in ice-cold o.g% NaCl and blotted on filter paper. Extraction Unless otherwise stated, all operations were performed at 4°. Dialysis was carried on until the desired pH and conductivity were reached. The tissue was cut into small pieces and homogenized in a Waring blender with o.g% NaCl (2l per kg wet weight). The homogenate was centrifuged at I4 ooo X g for 30 min. The precipitate was reextracted with o.g% N aCl (half the volume of the first supernatant), and the supernatants were combined. The extracts were concentrated by precipitation at 22° with (NH 4) 2S04 (2.5 M) and dialyzed against 25 mM Tris-HCl buffer (pH 7.0). To clear the extract it was centrifuged at I05 ooo X g for I8o min (Beckman Spinco L2-65 B, rotor 30). Chromatography on DEAE-cellulose The concentrated extract from a whole brain (about 350 ml) was applied to a column of DEAE-cellulose (Whatman DE 23) equilibrated with 25 mM Tris-HCl buffer (pH 7.0). Elution was performed with a linear salt gradient from 25 mM TrisHCl buffer (pH 7.0) to 25 mM Tris-HCl buffer (pH J.o) + I M NaCl at a constant rate of 2 ml· cm-2 · h-1 . The conductivity and the transmittance at 254 mft of the eluate were continuously recorded, and fractions of 20 ml were collected and assayed for butyrylcholinesterase activity. The butyrylcholinesterase Fractions A, B, C and D (Fig. IA) were concentrated about 40 times by dialysis against Carbowax 20M (Gurr), centrifuged and finally dialyzed against the buffer used in gel chromatography. Gel chromatography The chromatographic butyrylcholinesterase fractions were applied to a column of Sephadex G-200 (Pharmacia) equilibrated with 25 mM Tris-HCl buffer (pH 7.0) + I M NaCl. The column was eluted with the same buffer at a constant rate of 0.8 ml· cm- 2 ·h-I, and the transmittance at 254 mft was recorded continuously. Fractions of IO ml were collected and assayed for butyrylcholinesterase activity. Analytical gel chromatography was carried out on a column (go em x 5 cm 2 ) of Sephadex G-200 at a constant rate of 0.5 ml· cm- 2 • h-1 • The exclusion volume was determined with Blue Dextran 2000 (Pharmacia) and the molecular size estimated according to DETERMANN 5 •
Biochim. Biophys. Acta,
207
(1970) 477-484
MULTIPLE FORMS OF BUTYRYLCHOLINESTERASE
479
The butyrylcholinesterase Fractions I and II (Fig. rB) were concentrated by dialysis against Carbowax 20 M. Electrofocusing To avoid precipitation in the acid part of the column the samples were dialyzed against 5 mM sodium acetate buffer (pH 5-4) and centrifuged. The supernatant was dialyzed against a 0.5% solution of the carrier ampholyte to be used in the electrofocusing. Isoelectric focusing was carried out at 4 o in a rro-ml electro focusing column (LKB 8ror) 6 using r% solutions of Ampholine (LKB) as carrier ampholytes. The density gradient was prepared with sucrose (British Drug Houses Analar) by means of a gradient mixer (LKB 8r2r). The electrofocusing was continued for at least 6 h after the steady state was reached (20-roo h), as indicated by a constant current. The column was emptied at a rate of roo ml/h. Fractions of 2 ml were collected and assayed for butyrylcholinesterase activity, z8o-m,u absorbance and pH. The pH was determined at 22°. Determination of protein concentration In chromatography the transmittance at 254 m,u was recorded continuously (Uvicord I LKB, light path 3 mm). In the electrofocusing experiments the absorbance was measured at 280 m,u. In other samples (previously dialyzed against 25 mM sodium phosphate buffer (pH 7.0)) the protein concentration was estimated by the biuret reaction 7 with bovine serum albumin (Armour) as a standard (r mg/ml). Determination of butyrylcholinesterase activity Butyrylcholinesterase activity was determined spectrophotometrically8 : o. I ml of the sample was incubated at 30° in a thermostatted 3-ml cuvette (light path ro mm) with 3.0 ml of a solution of 0.093 M sodium phosphate buffer (pH 8.o), acetylcholinesterase-inhibitor 284c5rm * (0.33 ,uM) and 5,5-dithio-bis-(2-nitrobenzoic acid) (Aldrich) (0.33 mM). o.r ml of the substrate butyrylthiocholine iodide (Fluka) (o.ors M) was then added. In the blank, substrate was omitted. The change in absorbance at 412 m,u was recorded (Beckman DK 2A) and corrected for nonenzymatic hydrolysis of the substrate. Butyrylcholinesterase activity in turbid samples was determined as follows: 2.0 ml of the sample were incubated at 30° with 5·75 ml of a solution containing o.rzz M sodium phosphate buffer (pH 8.o) and acetylcholinesterase-inhibitor z84c5rm (0-43 ,uM). o.zs ml of the substrate butyrylthiocholine iodide (o.ors M) was then added. At intervals 8 separate aliquots of o.8 ml were taken out, and the reaction stopped by the addition of o.z ml of 0.76 M perchloric acid. The precipitate was removed by centrifugation, and o.8 ml of the supernatant was neutralized by addition of o.8 ml of 0.125 M NaOH. The thiocholine formed was determined by reading the absorbance at 412 m,u after the addition of o.os ml 5,5-dithio-bis-(2-nitrobenzoic acid) (o.or M). The two methods gave identical results with clear samples of butyrylcholinesterase.
* r,s-Bis-(4-allyldimethylammoniumphenyl)pentane-3-one diiodide (The Wellcome Research Laboratories).
Biochim. Biophys. Acta, 207 (r970) 477-484
J.
B. CARLSEN, 0. SVENSMARK
Treatment with neuraminidase (EC ].2.I.I8) Butyrylcholinesterase fractions dialyzed against 5 mM sodium phosphate buffer (pH 7.0) were mixed with an equal volume of buffer (o.r M sodium acetate, 0.2 M NaCl and 0.02 M CaC1 2 adjusted to pH 5·5 with 6 M HCl) and incubated for 48 hat 22° with o.or mg/ml of neuraminidase (Sigma Type VI). Another sample of the enzyme was treated in the same way but without addition ofneuraminidase.After incubation the samples were dialyzed against 5 mM Tris-HCl buffer (pH 7.0) and subsequently against a 0.5% solution of the carrier ampholyte used in the electrofocusing. TABLE I SPECIFIC ACTIVITIES OF BUTYRYLCHOLINESTERASE IN DIFFERENT FRACTIONS FROM HUMAN BRAIK
The values represent the mean of duplicate determinations on preparations obtained in a typical experiment.
Fraction
Butyrylcholineesterase ( flmoles fmin per mg protein)
Brain extract Precipitate from 2.5 M (NH 4 ) 2S0 4 Supernatant after ultracentrifugation Fractions obtained in DEAE-cellulose chromatography
0.007 o.ooS o.oog A o.o43 B o.oo6 c 0.003 D o.oo4 I o.r58
Fractions obtained in chromatography on Sephadex G-200 Supernatant after precipitation of Fraction I with acetic acid at pH 5-4 Supernatant after precipitation of Fraction II with acetic acid at pH 5·4
II O.OJ5
0.38I
0.033
RESULTS
Extraction of butyrylcholinesterase activity 41% (S.D. = 8%, n = I3) of the total butyrylcholinesterase activity in the brain tissue was extracted. In one experiment extraction was repeated until no more activity was released. The first extract contained 29%, the second 6%, the third I% and the fourth o% of the total butyrylcholinesterase activity in the homogenate. Attempts to solubilize the particle-bound butyrylcholinesterase Extraction of the tissue and of the freeze-dried and n-butanol-extracted tissue with NaCl in concentrations of o.I5 to 2.4 M and with o.I5 M sodium phosphate buffers at pH 6.o-8.5 did not increase the butyrylcholinesterase activity in the extract. Nor did homogenization of the tissue using the Potter-Elvehjem homogenizer or by ultrasonic treatment increase the yield. Treatment with detergents (r% Lubrol W, I% deoxycholate) and enzymes (trypsin, lecithinase D, neuraminidase) did not solubilize the enzyme. Biochim. Biophys. Acta, 207 (1970) 477-484
MULTIPLE FORMS OF BUTYRYLCHOLINESTERASE
B Conductivity
014 011
'E
-"
o.1o .E 008 0.06
:i: ~
:::1..
1254
40
FR.!
'"'!\ ExclUSIOn
60 0.01
vol.
80 100
l
FR.II 0.14 I
E
0 12}: 0 10 E
\
\
0.08
~
0.06
:1..
E
.04
600
Fig. r. Fractionation of human brain butyrylcholinesterase (BuChE). A. DEAE-cellulose chromatography of 300 rnl concentrated brain extract. Column size: 51 ern X 27.3 crn 2 • B. Gel chromatography on Sephadex G-200 of Fraction A. 14 ml of the concentrated sample were applied to the column (88 ern x rg.z crn 2 ).
Separation of butyrylcholinesterase fractions DEAE-cellulose chromatography. By DEAE-cellulose chromatography of the extract from human brain several fractions of butyrylcholinesterase activity were obtained (Fig. 1A). One inhomogeneous main fraction (A) was eluted at conductivities of 3-9 mS, two small fractions (Band C) from 9 to 13 mS and one fraction (D) at 17 mS. Identical results were obtained in 6 experiments with extracts from 6 brains. Gel chromatography. By gel chromatography on Sephadex G-200 Fraction A was completely separated into two inhomogeneous fractions, I and II (Fig. 1B). Similar results were obtained in 7 experiments with extracts from 6 brains. Fraction I appeared immediately after the exclusion volume, indicating a high molecular weight (300 000-400 ooo). Rechromatography on DEAE-cellulose gave two partially separated peaks at about 9 and 13 mS (22°). Fraction II was inhomogeneous and consisted of components of significantly lower molecular weight (5o ooo-150 ooo) than Fraction I. Rechromatography on DEAE-cellulose gave only one peak eluted at rr mS (22°). The butyrylcholinesterase activity of Fractions B and C was too small to allow gel chromatography. Fraction D appeared immediately after the exclusion volume on Sephadex G-200, indicating a high molecular weight. The specific activities of the butyrylcholinesterase fractions obtained in chromatography on DEAE-cellulose and Sephadex G-200 are given in Table I. Electrofocusing. Fraction I showed two main butyrylcholinesterase fractions, one focusing between pH 3.8 and 4·7 and one between 5.6 and 8.2 (Fig. 2A). Electrofocusing in narrower pH gradients resulted in further subfractionation: the acid fraction was separated into 7 and the neutral fraction into 9 subtractions. One of the samples contained in addition a subfraction at pH s.z. In analytical gel chromatography the two main fractions behaved as Fraction I. The relative amounts of the two fractions varied considerably in extracts from different brains: the acid fraction varied from 7 to s6% and the neutral fraction from 34 to go% of the total activity. In preparations in which the acid main fraction was small and the neutral fraction correspondingly large, the most acid subfractions were absent, and alkaline subfractions appeared with isoelectric points at 7·9 and 8.2. Biochim. Riophys. Acta. 207 (1970) 477-484
J. B. CARLSEN, 0. SVENSMARK
482 O.o7
A
fl,
~ 1, BuChE
' ~
pH10
.,., 06
'
>
6
o• 4
f
j
yJ
' \/\
I
~
\ i
I
'
8
0.06
o.os ''E
"\:
A:zeo pH 2D 10 1.6
0.03~
1.2
O.Q3.!!
o•
0.02
0
0.02
E
:>...
02 2
...
0.0 0
0.0 Fr~ction
.
o.o4 E
no.
~
::1..
0.01
fraction no.
50
Fig. 2. A. Electrofocusing of brain Fraction I. B. Electrofocusing of brain Fraction II. Abscissa: Number of fractions of 2.0 ml. BuChE, butyrylcholinesterase.
Electrofocusing of Fraction II revealed 4-6 subfractions in the pH range from 5.6 to 9.0 (Fig. 2B). Effect of neuraminidase To investigate whether butyrylcholinesterase fractions from human brain contain sialic acid residues, as does human serum butyrylcholinesterase9 , the effect of neuraminidase on the fractions was studied. The removal of sialic acid groups from human serum butyrylcholinesterase by neuraminidase results in a change in its isolectric point from about 3 to 7 (ref. ro). A similar change was observed after treatment with neuraminidase of the highmolecular-weight butyrylcholinesterase Fraction I obtained in gel chromatography. The amount of the acid main fraction decreased, and the neutral main fraction increased correspondingly (Fig. 3C and 3D). The most acid subfractions were lost, and alkaline subfractions appeared. Treatment of Fraction II with neuraminidase did not change the isoelectric point of the subfractions. Comparison with human serum butyrylcholinesterase In DEAE-cellulose chromatography human serum butyrylcholinesterase was eluted in one peak at 7·5 mS. In gel chromatography on Sephadex G-200 the enzyme was eluted in one peak appearing in the same volume as brain butyrylcholinesterase Fraction I 11 . Electrofocusing of serum resulted in several butyrylcholinesterase fractions with isoelectric points in the pH range from 3.8 to 4·5 (Fig. 3B). Treatment with neuraminidase caused a shift in the isoelectric point of the butyrylcholinesterase fractions: the acid subfractions disappeared completely, and alkaline subfractions appeared with isoelectric points from 7.1 to 8.r (Fig. 3A). DISCUSSION
The human cerebrum contains a large number of separable butyrylcholinesterase fractions. Chromatography on DEAE-cellulose showed one large inhomogeneous fraction and several minor fractions. In gel chromatography on Sephadex G-200 the main fraction was completely separated into two fractions. Electrofocusing of the high-molecular-weight Fraction I gave two main fractions, one acid (pH 3.8-4.7) and one neutral (pH 5.6-8.o), each exhibiting pronounced microheterogeneity. Also Biochim. Biophys. Acta, 207 (1970) 477-484
MULTIPLE FORMS OF BUTYRYLCHOLINESTERASE 9
10 0.14 0.10
SERUM
0.06 002
!..
0.14
~
0.10
SERUM
E
I
c
E oo6
4 pH
}\
3
NEURAMINIDASE TREATED
A
""'"'''~
~ 002 E
::>._
0.14 0.10
BRAIN
FRACTION 1
NEURAMINIDASE TREATED
c
0.06 0.02 0.14 0.10
0
0.06 0.02 10
4 pH
3
Fig. 3· Comparison between the butyrylcholinesterase patterns in electrofocusing of brain Fraction I and serum, untreated and neuraminidase treated. For each of the fractions collected from the column, the butyrylcholincsterase activity was plotted against pH at 22°.
Fraction II was separated into several subfractions in the pH range 5.6-8.0. Although definite reports on artifacts in electrofocusing are not available, the possibility that not all the subfractions represent real molecular species has to be taken into con~ sideration. Some of the acid subfraci.ions of the high~molecular-weight butyrylcholin~ esterase Fraction I are sialo-proteins as indicated by the change in the isoelectric points after treatment with neuraminidase. The human brain contains neuraminidase 12 , and although the pH optimum is about 4, neuraminidase may exert an effect on brain butyrylcholinesterase in vivo, after death or during the preparation. Hence, some of the alkaline subfractions of Fraction I may be a result of the action of neuraminidase. Similarly, the variation from preparation to preparation in the relative amounts of neutral and acid subfractions of Fraction I could be due to a more or less progressively degradative action of neuraminidase. The question arises whether butyrylcholinesterase found in the brain extract originated wholly or in part from plasma trapped in the brain tissue. The amount of soluble butyrylcholinesterase activity in rooo g of brain tissue corresponds to the amount present in 20-40 ml of plasma. Part of the plasma butyrylcholinesterase present in the brain extract must be expected to be precipitated by 2.5 M (NH 4) 2S04 (ref. I3) and to be present in the preparations used in this study. In DEAE-cellulose chromatography, plasma butyrylcholinesterase would be eluted together with Fraction A and in gel chromatography together with Fraction I. In electrofocusing, plasma butyrylcholinesterase showed about the same patterns as the acid main fraction Biochim. Biophys. Acta, 207 (r97o) 477-484
J. B. CARLSEN, 0. SVENSMARK obtained in electrofocusing Fraction I, and treatment with neuraminidase caused a shift in isoelectric point toward alkaline values (Fig. 3). Thus it remains uncertain whether the acid and the most alkaline subfractions of brain Fraction I derive from plasma, or whether they represent true components of the brain tissue. On the other hand, there are no indications that the remaining fractions originate from plasma. In conclusion, brain tissue contains (1) an insoluble butyrylcholinesterase fraction, (z) a soluble high-molecular-weight fraction consisting of 4-5 subfractions with isoelectric points from 5.6 to J.O, and (3) soluble fractions of lower molecular weight consisting of 3-4 fractions with isoelectric points from 5.6 to J.O, and 3 fractions with isoelectric points from 7·5 to 8.o. Furthermore, brain tissue may contain a highmolecular-weight fraction identical with the butyrylcholinesterase of plasma. The lowmolecular-weight subfractions with isoelectric points from 5.6 to J.O possibly represent subunits of the high-molecular-weight subfractions which focus in the same pH range. The existence of many different butyrylcholinesterase fractions in human brain made it impossible to prepare the isoenzymes in amounts allowing a detailed investigation of their enzymatic and molecular properties or of their distribution in the brain. Thus, at present no conclusions can be drawn as to the biological implications of the isoenzymes in brain. ACKNOWLEDGMENTS
This work represents a continuation of preliminary experiments carried out in this institute in 1964 by Ingrid M. Petersen, M.Sc. We are obliged to Mrs. Ellen Storm Hansen for skillful technical assistance and to the staff of the Institute of Forensic Medicine, University of Copenhagen, for their cooperation. The work was supported by grants from the Carlsberg Foundation and the Danish State Research Foundation. REFERENCES
1 0. SvENSMARK, Acta Physiol. Scand., 52 (rg6r) 372. W. GERHARDT, J. CLAUSEN, E. CHRISTENSEN AND J. RIISHEDE, Acta Neural. Scand., 39 (1963) 85. 3 D. J. EcoBICHON, Can. ]. Physiol. Pharmacal., 44 (rg66) 225. 4 K. D. BARRON AND J. BERNSOHN, j. Neurochem., 15 (rg68) 273. 5 H. DETERMANN, Gel Chromatography, Springer-Verlag, Berlin, rg68, p. uo. 6 0. VESTERBERG AND H. SvENSSON, Acta Chem. Scand., 20 (rg66) 820. 7 G. L. ELLMAN, Anal. Biochem., 3 (1962) 40. 8 G. L. ELLMAN, K. D. CouRTNEY, V. ANDRES, JR. AND R. M. FEATHERSTONE, Biochem. Pharmacal., 7 (rg6r) 88. 9 0. SvENSMARK, Acta Physiol. Scand., 52 (rg6r) 267. ro 0. SvENSMARK AND P. KRISTENSEN, Biochim. Biophys. Acta, 67 (1963) 441. II 0. SvENSMARK, Acta Physiol. Scand., 59 (1963) 378. 12 E. H. MoRGAN AND C. B. LAURELL, Nature, 197 (1963) 921. 13 0. SvENSMARK, Acta Physiol. Scand., 64, Suppl. 245 (1965) 13. 2
Biochim. Biophys. Acta, 207 (1970) 477-484
477
BBA 35576
MULTIPLE FORMS OF SOLUBLE BUTYRYLCHOLINESTERASE IN HUMAN BRAIN
JENS B. CARLSEN* AND OLE SVENSIVIARK* The Institute of Neurophysiology, University of Copenhagen, Copenhagen (Denmark) (Received December 2gth, rg6g)
SUMMARY r. 30-55% of the butyrylcholinesterase (EC 3.r.r.8) activity of human brain was present in an aqueous extract. Particle-bound butyrylcholinesterase could not be made soluble. 2. In DEAE-cellulose chromatography soluble butyrylcholinesterase was separated in one inhomogeneous main fraction and several minor fractions. Gel chromatography on Sephadex G-200 resolved the main fraction into two inhomogeneous fractions, one of higher molecular weight (300 000-400 ooo), the other of lower molecular weight (so ooo-rso ooo). 3· Electrofocusing of the butyrylcholinesterase fraction of higher molecular weight revealed two inhomogeneous fractions in the pH ranges 3.8-4.7 and 5.6-8.2. By treatment with neuraminidase (EC 3.2.r. r8) the most acid subfractions disappeared and alkaline subfractions appeared. The acid and the most alkaline subfractions may originate from plasma trapped in the tissue. Electrofocusing of the fraction of lower molecular weight showed at least 6 subfractions in the pH range 5.6-8.0. Treatment with neuraminidase had no effect on their isoelectric points. 4· Brain tissue thus contains at least 4-5 soluble butyrylcholinesterase fractions of high molecular weight with isoelectric points ranging from 5.6 to J.O and at least 6 low-molecular-weight fractions with isoelectric points from s.6 to 8.0.
INTRODUCTION In electrophoresis on paperl, agar gel2, and starch geP· 4 soluble butyrylcholinesterase activity from human brain occurred in 2-3 bands with different mobilities. Preliminary experiments (I. M. PETERSEN, unpublished) have shown that human brain butyrylcholinesterase could be separated in at least 3 fractions by DEAE* Present address: Department of Biochemistry C, University of Copenhagen, 71 Radmandsgade, DK 2200 Copenhagen N, Denmark.
Biochim. Biophys. Acta, 207 (r970) 477-484
J. B. CARLSEN, 0. SVENSMARK
cellulose chromatography and gel filtration. In the study presented in this report an attempt was made to isolate these fractions of butyrylcholinesterase in amounts allowing investigation of their properties, especially with respect to their content of sialic acid, and to compare them with human serum butyrylcholinesterase. Furthermore, an attempt was made to solubilize the particle-bound butyrylcholinesterase activity of human brain. MATERIALS AND METHODS Brain tissue IS human brains were obtained I-3 days after death. The causes of death were accidents (8), coronary occlusion (5), cancer colli uteri (I), and renal failure (I). The whole brain except for the cerebellum was used. After the removal of membranes and large vessels, the tissue was rinsed in ice-cold o.g% NaCl and blotted on filter paper. Extraction Unless otherwise stated, all operations were performed at 4°. Dialysis was carried on until the desired pH and conductivity were reached. The tissue was cut into small pieces and homogenized in a Waring blender with o.g% NaCl (2l per kg wet weight). The homogenate was centrifuged at I4 ooo X g for 30 min. The precipitate was reextracted with o.g% N aCl (half the volume of the first supernatant), and the supernatants were combined. The extracts were concentrated by precipitation at 22° with (NH 4) 2S04 (2.5 M) and dialyzed against 25 mM Tris-HCl buffer (pH 7.0). To clear the extract it was centrifuged at I05 ooo X g for I8o min (Beckman Spinco L2-65 B, rotor 30). Chromatography on DEAE-cellulose The concentrated extract from a whole brain (about 350 ml) was applied to a column of DEAE-cellulose (Whatman DE 23) equilibrated with 25 mM Tris-HCl buffer (pH 7.0). Elution was performed with a linear salt gradient from 25 mM TrisHCl buffer (pH 7.0) to 25 mM Tris-HCl buffer (pH J.o) + I M NaCl at a constant rate of 2 ml· cm-2 · h-1 . The conductivity and the transmittance at 254 mft of the eluate were continuously recorded, and fractions of 20 ml were collected and assayed for butyrylcholinesterase activity. The butyrylcholinesterase Fractions A, B, C and D (Fig. IA) were concentrated about 40 times by dialysis against Carbowax 20M (Gurr), centrifuged and finally dialyzed against the buffer used in gel chromatography. Gel chromatography The chromatographic butyrylcholinesterase fractions were applied to a column of Sephadex G-200 (Pharmacia) equilibrated with 25 mM Tris-HCl buffer (pH 7.0) + I M NaCl. The column was eluted with the same buffer at a constant rate of 0.8 ml· cm- 2 ·h-I, and the transmittance at 254 mft was recorded continuously. Fractions of IO ml were collected and assayed for butyrylcholinesterase activity. Analytical gel chromatography was carried out on a column (go em x 5 cm 2 ) of Sephadex G-200 at a constant rate of 0.5 ml· cm- 2 • h-1 • The exclusion volume was determined with Blue Dextran 2000 (Pharmacia) and the molecular size estimated according to DETERMANN 5 •
Biochim. Biophys. Acta,
207
(1970) 477-484
MULTIPLE FORMS OF BUTYRYLCHOLINESTERASE
479
The butyrylcholinesterase Fractions I and II (Fig. rB) were concentrated by dialysis against Carbowax 20 M. Electrofocusing To avoid precipitation in the acid part of the column the samples were dialyzed against 5 mM sodium acetate buffer (pH 5-4) and centrifuged. The supernatant was dialyzed against a 0.5% solution of the carrier ampholyte to be used in the electrofocusing. Isoelectric focusing was carried out at 4 o in a rro-ml electro focusing column (LKB 8ror) 6 using r% solutions of Ampholine (LKB) as carrier ampholytes. The density gradient was prepared with sucrose (British Drug Houses Analar) by means of a gradient mixer (LKB 8r2r). The electrofocusing was continued for at least 6 h after the steady state was reached (20-roo h), as indicated by a constant current. The column was emptied at a rate of roo ml/h. Fractions of 2 ml were collected and assayed for butyrylcholinesterase activity, z8o-m,u absorbance and pH. The pH was determined at 22°. Determination of protein concentration In chromatography the transmittance at 254 m,u was recorded continuously (Uvicord I LKB, light path 3 mm). In the electrofocusing experiments the absorbance was measured at 280 m,u. In other samples (previously dialyzed against 25 mM sodium phosphate buffer (pH 7.0)) the protein concentration was estimated by the biuret reaction 7 with bovine serum albumin (Armour) as a standard (r mg/ml). Determination of butyrylcholinesterase activity Butyrylcholinesterase activity was determined spectrophotometrically8 : o. I ml of the sample was incubated at 30° in a thermostatted 3-ml cuvette (light path ro mm) with 3.0 ml of a solution of 0.093 M sodium phosphate buffer (pH 8.o), acetylcholinesterase-inhibitor 284c5rm * (0.33 ,uM) and 5,5-dithio-bis-(2-nitrobenzoic acid) (Aldrich) (0.33 mM). o.r ml of the substrate butyrylthiocholine iodide (Fluka) (o.ors M) was then added. In the blank, substrate was omitted. The change in absorbance at 412 m,u was recorded (Beckman DK 2A) and corrected for nonenzymatic hydrolysis of the substrate. Butyrylcholinesterase activity in turbid samples was determined as follows: 2.0 ml of the sample were incubated at 30° with 5·75 ml of a solution containing o.rzz M sodium phosphate buffer (pH 8.o) and acetylcholinesterase-inhibitor z84c5rm (0-43 ,uM). o.zs ml of the substrate butyrylthiocholine iodide (o.ors M) was then added. At intervals 8 separate aliquots of o.8 ml were taken out, and the reaction stopped by the addition of o.z ml of 0.76 M perchloric acid. The precipitate was removed by centrifugation, and o.8 ml of the supernatant was neutralized by addition of o.8 ml of 0.125 M NaOH. The thiocholine formed was determined by reading the absorbance at 412 m,u after the addition of o.os ml 5,5-dithio-bis-(2-nitrobenzoic acid) (o.or M). The two methods gave identical results with clear samples of butyrylcholinesterase.
* r,s-Bis-(4-allyldimethylammoniumphenyl)pentane-3-one diiodide (The Wellcome Research Laboratories).
Biochim. Biophys. Acta, 207 (r970) 477-484
J.
B. CARLSEN, 0. SVENSMARK
Treatment with neuraminidase (EC ].2.I.I8) Butyrylcholinesterase fractions dialyzed against 5 mM sodium phosphate buffer (pH 7.0) were mixed with an equal volume of buffer (o.r M sodium acetate, 0.2 M NaCl and 0.02 M CaC1 2 adjusted to pH 5·5 with 6 M HCl) and incubated for 48 hat 22° with o.or mg/ml of neuraminidase (Sigma Type VI). Another sample of the enzyme was treated in the same way but without addition ofneuraminidase.After incubation the samples were dialyzed against 5 mM Tris-HCl buffer (pH 7.0) and subsequently against a 0.5% solution of the carrier ampholyte used in the electrofocusing. TABLE I SPECIFIC ACTIVITIES OF BUTYRYLCHOLINESTERASE IN DIFFERENT FRACTIONS FROM HUMAN BRAIK
The values represent the mean of duplicate determinations on preparations obtained in a typical experiment.
Fraction
Butyrylcholineesterase ( flmoles fmin per mg protein)
Brain extract Precipitate from 2.5 M (NH 4 ) 2S0 4 Supernatant after ultracentrifugation Fractions obtained in DEAE-cellulose chromatography
0.007 o.ooS o.oog A o.o43 B o.oo6 c 0.003 D o.oo4 I o.r58
Fractions obtained in chromatography on Sephadex G-200 Supernatant after precipitation of Fraction I with acetic acid at pH 5-4 Supernatant after precipitation of Fraction II with acetic acid at pH 5·4
II O.OJ5
0.38I
0.033
RESULTS
Extraction of butyrylcholinesterase activity 41% (S.D. = 8%, n = I3) of the total butyrylcholinesterase activity in the brain tissue was extracted. In one experiment extraction was repeated until no more activity was released. The first extract contained 29%, the second 6%, the third I% and the fourth o% of the total butyrylcholinesterase activity in the homogenate. Attempts to solubilize the particle-bound butyrylcholinesterase Extraction of the tissue and of the freeze-dried and n-butanol-extracted tissue with NaCl in concentrations of o.I5 to 2.4 M and with o.I5 M sodium phosphate buffers at pH 6.o-8.5 did not increase the butyrylcholinesterase activity in the extract. Nor did homogenization of the tissue using the Potter-Elvehjem homogenizer or by ultrasonic treatment increase the yield. Treatment with detergents (r% Lubrol W, I% deoxycholate) and enzymes (trypsin, lecithinase D, neuraminidase) did not solubilize the enzyme. Biochim. Biophys. Acta, 207 (1970) 477-484
MULTIPLE FORMS OF BUTYRYLCHOLINESTERASE
B Conductivity
014 011
'E
-"
o.1o .E 008 0.06
:i: ~
:::1..
1254
40
FR.!
'"'!\ ExclUSIOn
60 0.01
vol.
80 100
l
FR.II 0.14 I
E
0 12}: 0 10 E
\
\
0.08
~
0.06
:1..
E
.04
600
Fig. r. Fractionation of human brain butyrylcholinesterase (BuChE). A. DEAE-cellulose chromatography of 300 rnl concentrated brain extract. Column size: 51 ern X 27.3 crn 2 • B. Gel chromatography on Sephadex G-200 of Fraction A. 14 ml of the concentrated sample were applied to the column (88 ern x rg.z crn 2 ).
Separation of butyrylcholinesterase fractions DEAE-cellulose chromatography. By DEAE-cellulose chromatography of the extract from human brain several fractions of butyrylcholinesterase activity were obtained (Fig. 1A). One inhomogeneous main fraction (A) was eluted at conductivities of 3-9 mS, two small fractions (Band C) from 9 to 13 mS and one fraction (D) at 17 mS. Identical results were obtained in 6 experiments with extracts from 6 brains. Gel chromatography. By gel chromatography on Sephadex G-200 Fraction A was completely separated into two inhomogeneous fractions, I and II (Fig. 1B). Similar results were obtained in 7 experiments with extracts from 6 brains. Fraction I appeared immediately after the exclusion volume, indicating a high molecular weight (300 000-400 ooo). Rechromatography on DEAE-cellulose gave two partially separated peaks at about 9 and 13 mS (22°). Fraction II was inhomogeneous and consisted of components of significantly lower molecular weight (5o ooo-150 ooo) than Fraction I. Rechromatography on DEAE-cellulose gave only one peak eluted at rr mS (22°). The butyrylcholinesterase activity of Fractions B and C was too small to allow gel chromatography. Fraction D appeared immediately after the exclusion volume on Sephadex G-200, indicating a high molecular weight. The specific activities of the butyrylcholinesterase fractions obtained in chromatography on DEAE-cellulose and Sephadex G-200 are given in Table I. Electrofocusing. Fraction I showed two main butyrylcholinesterase fractions, one focusing between pH 3.8 and 4·7 and one between 5.6 and 8.2 (Fig. 2A). Electrofocusing in narrower pH gradients resulted in further subfractionation: the acid fraction was separated into 7 and the neutral fraction into 9 subtractions. One of the samples contained in addition a subfraction at pH s.z. In analytical gel chromatography the two main fractions behaved as Fraction I. The relative amounts of the two fractions varied considerably in extracts from different brains: the acid fraction varied from 7 to s6% and the neutral fraction from 34 to go% of the total activity. In preparations in which the acid main fraction was small and the neutral fraction correspondingly large, the most acid subfractions were absent, and alkaline subfractions appeared with isoelectric points at 7·9 and 8.2. Biochim. Riophys. Acta. 207 (1970) 477-484
J. B. CARLSEN, 0. SVENSMARK
482 O.o7
A
fl,
~ 1, BuChE
' ~
pH10
.,., 06
'
>
6
o• 4
f
j
yJ
' \/\
I
~
\ i
I
'
8
0.06
o.os ''E
"\:
A:zeo pH 2D 10 1.6
0.03~
1.2
O.Q3.!!
o•
0.02
0
0.02
E
:>...
02 2
...
0.0 0
0.0 Fr~ction
.
o.o4 E
no.
~
::1..
0.01
fraction no.
50
Fig. 2. A. Electrofocusing of brain Fraction I. B. Electrofocusing of brain Fraction II. Abscissa: Number of fractions of 2.0 ml. BuChE, butyrylcholinesterase.
Electrofocusing of Fraction II revealed 4-6 subfractions in the pH range from 5.6 to 9.0 (Fig. 2B). Effect of neuraminidase To investigate whether butyrylcholinesterase fractions from human brain contain sialic acid residues, as does human serum butyrylcholinesterase9 , the effect of neuraminidase on the fractions was studied. The removal of sialic acid groups from human serum butyrylcholinesterase by neuraminidase results in a change in its isolectric point from about 3 to 7 (ref. ro). A similar change was observed after treatment with neuraminidase of the highmolecular-weight butyrylcholinesterase Fraction I obtained in gel chromatography. The amount of the acid main fraction decreased, and the neutral main fraction increased correspondingly (Fig. 3C and 3D). The most acid subfractions were lost, and alkaline subfractions appeared. Treatment of Fraction II with neuraminidase did not change the isoelectric point of the subfractions. Comparison with human serum butyrylcholinesterase In DEAE-cellulose chromatography human serum butyrylcholinesterase was eluted in one peak at 7·5 mS. In gel chromatography on Sephadex G-200 the enzyme was eluted in one peak appearing in the same volume as brain butyrylcholinesterase Fraction I 11 . Electrofocusing of serum resulted in several butyrylcholinesterase fractions with isoelectric points in the pH range from 3.8 to 4·5 (Fig. 3B). Treatment with neuraminidase caused a shift in the isoelectric point of the butyrylcholinesterase fractions: the acid subfractions disappeared completely, and alkaline subfractions appeared with isoelectric points from 7.1 to 8.r (Fig. 3A). DISCUSSION
The human cerebrum contains a large number of separable butyrylcholinesterase fractions. Chromatography on DEAE-cellulose showed one large inhomogeneous fraction and several minor fractions. In gel chromatography on Sephadex G-200 the main fraction was completely separated into two fractions. Electrofocusing of the high-molecular-weight Fraction I gave two main fractions, one acid (pH 3.8-4.7) and one neutral (pH 5.6-8.o), each exhibiting pronounced microheterogeneity. Also Biochim. Biophys. Acta, 207 (1970) 477-484
MULTIPLE FORMS OF BUTYRYLCHOLINESTERASE 9
10 0.14 0.10
SERUM
0.06 002
!..
0.14
~
0.10
SERUM
E
I
c
E oo6
4 pH
}\
3
NEURAMINIDASE TREATED
A
""'"'''~
~ 002 E
::>._
0.14 0.10
BRAIN
FRACTION 1
NEURAMINIDASE TREATED
c
0.06 0.02 0.14 0.10
0
0.06 0.02 10
4 pH
3
Fig. 3· Comparison between the butyrylcholinesterase patterns in electrofocusing of brain Fraction I and serum, untreated and neuraminidase treated. For each of the fractions collected from the column, the butyrylcholincsterase activity was plotted against pH at 22°.
Fraction II was separated into several subfractions in the pH range 5.6-8.0. Although definite reports on artifacts in electrofocusing are not available, the possibility that not all the subfractions represent real molecular species has to be taken into con~ sideration. Some of the acid subfraci.ions of the high~molecular-weight butyrylcholin~ esterase Fraction I are sialo-proteins as indicated by the change in the isoelectric points after treatment with neuraminidase. The human brain contains neuraminidase 12 , and although the pH optimum is about 4, neuraminidase may exert an effect on brain butyrylcholinesterase in vivo, after death or during the preparation. Hence, some of the alkaline subfractions of Fraction I may be a result of the action of neuraminidase. Similarly, the variation from preparation to preparation in the relative amounts of neutral and acid subfractions of Fraction I could be due to a more or less progressively degradative action of neuraminidase. The question arises whether butyrylcholinesterase found in the brain extract originated wholly or in part from plasma trapped in the brain tissue. The amount of soluble butyrylcholinesterase activity in rooo g of brain tissue corresponds to the amount present in 20-40 ml of plasma. Part of the plasma butyrylcholinesterase present in the brain extract must be expected to be precipitated by 2.5 M (NH 4) 2S04 (ref. I3) and to be present in the preparations used in this study. In DEAE-cellulose chromatography, plasma butyrylcholinesterase would be eluted together with Fraction A and in gel chromatography together with Fraction I. In electrofocusing, plasma butyrylcholinesterase showed about the same patterns as the acid main fraction Biochim. Biophys. Acta, 207 (r97o) 477-484
J. B. CARLSEN, 0. SVENSMARK obtained in electrofocusing Fraction I, and treatment with neuraminidase caused a shift in isoelectric point toward alkaline values (Fig. 3). Thus it remains uncertain whether the acid and the most alkaline subfractions of brain Fraction I derive from plasma, or whether they represent true components of the brain tissue. On the other hand, there are no indications that the remaining fractions originate from plasma. In conclusion, brain tissue contains (1) an insoluble butyrylcholinesterase fraction, (z) a soluble high-molecular-weight fraction consisting of 4-5 subfractions with isoelectric points from 5.6 to J.O, and (3) soluble fractions of lower molecular weight consisting of 3-4 fractions with isoelectric points from 5.6 to J.O, and 3 fractions with isoelectric points from 7·5 to 8.o. Furthermore, brain tissue may contain a highmolecular-weight fraction identical with the butyrylcholinesterase of plasma. The lowmolecular-weight subfractions with isoelectric points from 5.6 to J.O possibly represent subunits of the high-molecular-weight subfractions which focus in the same pH range. The existence of many different butyrylcholinesterase fractions in human brain made it impossible to prepare the isoenzymes in amounts allowing a detailed investigation of their enzymatic and molecular properties or of their distribution in the brain. Thus, at present no conclusions can be drawn as to the biological implications of the isoenzymes in brain. ACKNOWLEDGMENTS
This work represents a continuation of preliminary experiments carried out in this institute in 1964 by Ingrid M. Petersen, M.Sc. We are obliged to Mrs. Ellen Storm Hansen for skillful technical assistance and to the staff of the Institute of Forensic Medicine, University of Copenhagen, for their cooperation. The work was supported by grants from the Carlsberg Foundation and the Danish State Research Foundation. REFERENCES
1 0. SvENSMARK, Acta Physiol. Scand., 52 (rg6r) 372. W. GERHARDT, J. CLAUSEN, E. CHRISTENSEN AND J. RIISHEDE, Acta Neural. Scand., 39 (1963) 85. 3 D. J. EcoBICHON, Can. ]. Physiol. Pharmacal., 44 (rg66) 225. 4 K. D. BARRON AND J. BERNSOHN, j. Neurochem., 15 (rg68) 273. 5 H. DETERMANN, Gel Chromatography, Springer-Verlag, Berlin, rg68, p. uo. 6 0. VESTERBERG AND H. SvENSSON, Acta Chem. Scand., 20 (rg66) 820. 7 G. L. ELLMAN, Anal. Biochem., 3 (1962) 40. 8 G. L. ELLMAN, K. D. CouRTNEY, V. ANDRES, JR. AND R. M. FEATHERSTONE, Biochem. Pharmacal., 7 (rg6r) 88. 9 0. SvENSMARK, Acta Physiol. Scand., 52 (rg6r) 267. ro 0. SvENSMARK AND P. KRISTENSEN, Biochim. Biophys. Acta, 67 (1963) 441. II 0. SvENSMARK, Acta Physiol. Scand., 59 (1963) 378. 12 E. H. MoRGAN AND C. B. LAURELL, Nature, 197 (1963) 921. 13 0. SvENSMARK, Acta Physiol. Scand., 64, Suppl. 245 (1965) 13. 2
Biochim. Biophys. Acta, 207 (1970) 477-484