Human erythrocyte acetylcholinesterase I. Resolution of activity into two components

612

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 3 5 8 6 9

HUMAN E R Y T H R O C Y T E A C E T Y L C H O L I N E S T E R A S E I. RESOLUTION OF ACTIVITY INTO TWO COMPONENTS

T ( ) U R A J S H A F A I AND J E A N A. C O I ~ T N E R

Department of Pediatrics, State University of New York at Buffalo, and Buffalo Children's Hospital, Bl*ffalo, N . Y . (U.S.A.) (Received D e c e m b e r 2nd, 197 o)

SUMMARY

I. Human erythrocyte acetylcholinesterase solubilized by Triton X-Ioo has been resolved into two components by DEAE-Sephadex column chromatography. The two partially purified enzymes have apparently identical molecular weights with different electrophoretic mobility. The Km values of the two components were shown not to differ significantly. 2. The molecular weight of Triton-solubilized acetylcholinesterase was estimated by gel filtration, in the presence of Triton X-Ioo, to be approx. 42o ooo. Following the removal of the detergent, the molecular weight of the enzyme was shown to be greater than 2" lO6.

INTRODUCTION

Mammalian erythrocyte acetylcholinesterase(s) have not been well characterized. The purification of the human 1 and bovine 2 erythrocyte acetylcholinesterase have been reported. However, the molecular weight, sedimentation and diffusion constants and other properties of these purified enzymes have not been determined. The possibility of the presence of two acetylcholinesterases or even a mixture of true and pseudocholinesterase in the erythrocytes was also considered by some investigatorsa, 4. The present work was undertaken to study the possibility of the heterogeneity of this enzyme in the erythrocytes, as well as the determination of the molecular weight of the partially purified enzyme by gel filtration. The effect of the detergent, Triton X-Ioo, on the molecular size of the acetylcholinesterase was also investigated. MATERIALS

All reagents were of analytical grade. Agarose (Sepharose 4 B) and DEAESephadex (A-5o) were obtained from Pharmacia (Uppsala, Sweden). Cellulose acetate Biochim. Biophys. Acta, 236 (1971) 612-618

HUMAN ERYTHROCYTE ACETYLCHOLINESTERASE

613

gel (Cellogel), o.3 m m thickness, was from Chemetron (Milan, Italy) and Triton X - I o o from R o h m & Haas (Philadelphia, Pa.). METHODS

ASSayS Acetylcholinesterase activity was determined colorimetrically ~, using a Beckman DU spectrophotometer equipped with a recorder. The protein concentration was determined by the method of LowRY et al. 6 with bovine serum albumin (Sigma Chemical Co.) as standard.

Preparation of Triton-solubilized stromal acetylcholinesterase Heparinized blood (20 4 ° ml) was obtained from h u m a n volunteers. The hemoglobin-free s t r o m a t a were prepared according to the method of DODGE et ald from the freshly drawn blood. An equal volume of 5 % Triton X - I o o (in phosphate buffer*, p H 8.0, o.oi M) was then added to the packed stromata and centrifuged at I o ooo × g for 0.5 h at 4 °. The clear supernatant was then subjected to one or more of the following procedures: I. D E A E-Sephadex column chromatography of Triton-solubilized acetylcholinesterase. D E A E - S e p h a d e x (A-5o) previously washed with HC1 (I M) and N a O H (I M) was equilibrated with 0.05% Triton X - I o o in phosphate buffer (pH 8, o.oi M). It was poured into a column (15 cm × 1. 5 cm) and equilibrated with the same buffer (IOO ml). Following the application of the Triton-solubilized acetylcholinesterase, the column was eluted with IOO ml of the above buffer followed by a linear salt gradient obtained b y adding in an open chamber: 200 ml of 0.75 M NaC1 (in phosphate buffer, p H 8, o.oi M, o . o 5 yo Triton X-ioo) to 200 ml of phosphate buffer (pH 8, o.oi M, 0.05% Triton X-Ioo). The flow rate was approximately IO ml/h and 5-ml fractions were collected. The fractions containing acetylcholinesterase activity were pooled, dialyzed (against several changes of Tris buffer, p H 7.2, 0.05 M) and concentrated in membrane filters of less than 5 m/z porosity. 2. Gel-filtration of acetylcholincsterase. Agarose (Sepharose 4B) equilibrated with Tris buffer (pH 7.2, 0.05 M, 0.05% Triton X-Ioo) was poured into a column (9 ° c m x 2 era). Following equilibration of the column at 4 o with 400 ml of the same buffer, it was calibrated with thyroglobulin (tool. wt. : 669 ooo), beef liver catalase (tool. wt. : 247 ooo) and yeast alcohol dehydrogenase (tool. wt. : 15o ooo). The apparent molecular weight of acetylcholinesterase was determined according to the method of ACKERSs. In all the experiments the flow rate was approximately 6 ml/h and 2-ml fractions were collected. 3. Butanol extraction of Triton-solubilized acetyleholinesterase. Triton-solubilized acetylcholinesterase was lyophilized and the residue extracted twice with butanol (IO ml), followed b y extraction with anhydrous ether (IO ml), both at room temperature. The residue was then dried in vacuo, taken up in Tris buffer (pH 7.2, 0.05 M) and dispersed by sonication for 5-1o sec at 4 ° ooo oscillations/sec. The suspension was then centrifuged at IO ooo × g for 0.5 h and the clear supernatant collected. 4. Electrophoresis. Electrophoresis on cellulose acetate gel was performed in a * All buffers used were I mM in respect to F.DTA and 2-mercaptoethanol.

Biochim. Biophys. dcta, 236 (1971) 012 618

6i 4

T. SHAFAI. J. A. CORTNER

Shandon electrophoresis chamber (Shandon Scientific Co., Sewickley, Pa.) at 4L After equilibration of the gel with veronal buffer (pH 8.6, 0.o25 M), preparations of acetylcholinesterase (2 5 ffl, containing approx. 2O-lOO fig protein) were applied at the origin and run for i h at IO V/cm. The gels were stained by a modification of the method of KARNOFSKY AND ROOTS'a. To 13 ml of acetate buffer (pH 7.8, 0.5 M), 5o mg acetylthiocholine iodide, I ml sodium citrate (I M), 4 ml CuSO 4 (I. 5 M) and 2 ml KaFe(CN)6 (o.o5 M) were added in that order, mixed, poured on the gel and incubated at 37 '~ for 5-3o rain. RESULTS

Agarose gel filtration of Triton-solubilized acetylcholinesterase in the presence of Triton X-Ioo revealed a single peak of enzymatic activity with an apparent molecular weight of 42o ooo (Figs. IA and 2). When the non-ionic detergent, Triton

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Fig. I. A. G e l - f i l t r a t i o n p a t t e r n of a c e t y l c h o l i n e s t e r a s e o n 4 °4, a g a r o s e ; c o l u n m e q u i l i b r a t e d w i t h T r i t o n X - i o o (0.05 %). A c e t y l c h o l i n e s t e r a s e w a s f i l t e r e d t h r o u g h a c o l u m n of 40/o a g a r o s e . T h e v o i d v o l u m e of t h e c o l u m n w a s d e t e r m i n e d w i t h B l u e D e x t r a n 20oo a n d s h o w n t o b e r e l a t i v e l y c o n s t a n t ( i 2 ml) in s e v e r a l e x p e r i m e n t s . I n c r e a s i n g t h e c o n c e n t r a t i o n of T r i t o n X - I o o t o I o.~ d i d n o t a l t e r t h e e l u t i o n p a t t e r n . 13. G e l - f i l t r a t i o n p a t t e r n of a c e t y l c h o l i n e s t e r a s e o n 4 °'o a g a r o s e ; column equilibrated with buffer and without Triton X-Ioo. Butanol extracted acetylcholine s t e r a s e p r e p a r a t i o n w a s f i l t e r e d t h r o u g h a c o l u m n o f 4 % a g a r o s e as d e s c r i b e d in METHODS. T h e v o i d v o l m n e w a s d e t e r m i n e d w i t h B l u e D e x t r a n 2ooo a n d s h o w n t o b e r e l a t i v e l y c o n s t a n t ( ± 2 m l ) . T h e e l u t i o n v o l u m e of t h y r o g l o b u l i n , b e e f l i v e r c a t a l a s e a n d y e a s t a l c o h o l d e h y d r o g e n a s e w e r e t h e s a m e as b e f o r e ( ± 2 ml). N o c o r r e c t i o n s w e r e m a d e for t h e c o n t r i b u t i o n of t u r b i d i t y t o t h e a b s o r b a n c e a t 280 mff of t h e f r a c t i o n s c o n t a i n i n g a c e t y l c h o l i n e s t e r a s e a c t i v i t y . T h e r e c o v e r y of t h e e n z y m a t i c a c t i v i t y w a s a p p r o x . 6o % .

Biochim. Biophys. Mcta, 236 (1971) 6 1 2 - 6 1 8

HUMAN ERYTHROCYTE ACETYLCHOLINESTERASE

615

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Fig. 2. Calibration line of 4 % agarose colunln. The points represent average Ve/V o values for (left to right) : thyroglobulin (mol. wt. : 669 ooo), beef liver catalase (tool. wt, : 247 ooo) and y e a s t alcohol dehydrogenase (moI. wt. : 15o ooo), from five experiments. Arrows: Ve/V o and estimated molecular weight of e r y t h r o c y t e acetylcholinesterase (average of five experiments).

X-Ioo, was removed from the Triton-solubilized acetylcholinesterase by butanolether extraction (approximate recovery 6o-7o%) and the preparation subjected to gel filtration on the same column equilibrated with Tris buffer (in the absence of Triton X-Ioo), all of the enzymatic activity was eluted with the void volume, indicating a molecular weight greater than 2. lO 6 (Fig. IB). Agarose gel filtration of the butanol-ether extracted acetylcholinesterase preparation on the same column equilibrated with Triton X-ioo (o.o5% in Tris buffer) revealed an elution pattern similar to that shown in Fig. IA, indicating an apparent molecular weight of 42o ooo.

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Fig. 3- D E A E - S e p h a d e x column c h r o m a t o g r a p h y p a t t e r n of acetvlcholinesterase. Tritonsolubilizcd p r e p a r a t i o n was c h r o m a t o g r a p h e d on a column of D E A F - S e p h a d e x as described in METHODS. The recovery of enzymatic activity was approx. 7oO~,.

Biochim. Biophys. Acta, 236 (1971 ) 612-618

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T. SHAFAI, J. A. CORTNER

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I"ig, 4. D E A E - S e p h a d e x column c h r o m a t o g r a p h y p a t t e r n s of acetylcholinesterase-i and acetylcholinesterase-2. P r e p a r a t i o n s of acetylcholinesterase-i and acetylcholinesterase-2 were rechrom a t o g r a p h e d on a column of D E A E - S e p h a d e x as described in METHODS.

DEAE-Sephadex column chromatography of Triton-solubilized acetylcholinesterase demonstrated the presence of two peaks of enzymatic activity (Fig. 3) which were eluted at NaC1 concentrations of o.15 M (acetylcholinesterase-I) and o.25 M (acetylcholinesterase-2). Fractions containing acetylcholinesterase activity (acetylcholinesterase-I, 6o-IiO ml and acetylcholinesterase-2, 155-2oo ml) were dialyzed and reehromatographed separately on DEAE-Sephadex, under identical conditions. Both components maintained their respective position, being eluted as single peaks of acetylcholinesterase activity at NaC1 concentrations of o.15 and o.25 M (Fig. 4). Cellulose acetate gel electrophoresis of the two components showed that acetylcholinesterase-2 migrated more rapidly towards the anode (Fig. 5). Electrophoresis of the crude Triton-solubilized acetylcholinesterase (o.5-2.5} ~ Triton), on the other hand, revealed considerable streaking of the enzymes with most of the enzymatic activity remaining at the point of application*. Agarose gel filtration of both enzymes, in the presence of Triton X-Ioo, revealed single peaks of enzymatic activity with an apparent molecular weight of 420 ooo in each case. Preparations of acetylcholinesterase-I and acetylcholinesterase-2 from DEAESephadex column of approx. 2oo- and 5o-fold purification, respectively, were used for the following studies. When eserine sulfate was added to the enzymes before the addition of substrate, it was shown to be a non-competitive inhibitor of both enzymes with a K~ value of 9" 1°-9 M for both enzymes. Similarly, di-isopropyl fluorophosphate (DFP) was shown to be a non-competitive inhibitor of both acetylcholinLower concentrations of Triton X-I oo did not solubilize appreciable quantities of acetylcholinestesterase from the stromata.

Biochim. Biophys. Acta, 236 (t97 I) 612-618

HUMAN ERYTHROCYTE ACETYLCHOLINESTERASE

617

Fig. 5. Cellulose a c e t a t e gel electrophoresis of acetylcholinesterase-I a n d acetylcholinesterase-2. Aliquots of (left to right) a c e t y l c h o l i n e s t e r a s e - I , acetylcholine sterase-2 a n d a m i x t u r e of t h e two c o m p o n e n t s were electrophoresed a n d s t a i n e d as described in METHODS. T h e origin was m a r k e d b y P o l y n k (George T. Gurr, Ltd., L o n d o n , England) before application of t h e samples.

esterases with a K i of approx. 1.2.IO 7 M. Quinidine sulfate was not i n h i b i t o r y to either of these enzymes at a concentration of I .lO .5 M, which would inhibit the serum pseudocholinesterase a°. The Michaelis c o n s t a n t s for acetylthiocholine iodide of acetylcholinesterase-I a n d acetylcholinesterase-2 d e t e r m i n e d with s u b s t r a t e concentrations ranging from o.125 • IO 4 to 12.5" IO 4 were found to be 1.25" IO 4 M a n d 1.7 "lO-4 M, respectively• S u b s t r a t e inhibition at concentrations up to 25 • i o 2 M of acetylthiocholine iodide was not observed for either of these enzymes.

DISCUSSION

The results of agarose gel filtration of acetylcholinesterase with a n d w i t h o u t the non-iomc detergent, T r i t o n X - i o o , can be best e x p l a i n e d b y aggregation of the e n z y m e in the absence of detergent. A similar p h e n o m e n o n was o b s e r v e d b y CHANGEUX et al. al with purified eel electroplax acetylcholinesterase. The existence of this aggregation u n d e r physiological conditions is in doubt. Since it is known t h a t the purified eel electroplax acetylcholinesterase does not form large aggregates spont a n e o u s l y n , ~2, it is possible t h a t the aggregation of the red cell e n z y m e in the absence of Triton X - I o o m a y be due, at least in p a r t , to non-polar interactions between m e m b r a n e proteins a n d acetylcholinesterase. Biochim. Biophys. Acla, 236 (i97i) 6t2 618

618

T. SHAFAI, J. A. CORTNER

The molecular weight of 420 000 estimated by gel filtration in the absence of Triton X-Ioo is in disagreement with tile recent results of BELLHORN et al. 13. The difference in the method of solubilization of tile enzyme from tile stromata might account for the observed difference in the estimated molecular weights. The nature and significance of the heterogeneity of the erythrocyte acetylcholinesterase are not clear for the time being. Although the two components differ in charge as demonstrated by ion exchange chromatography and by electrophoresis, they have apparently identical molecular weights. The Km values of the two components are not significantly different and are similar to those reported for the purified brain acetylcholinesterase preparations14,15. Further purification and characterization as well as detailed kinetic studies of tile two components are in progress. ACKNOWLEDGEMENTS

Dr. Shafai is a Postdoctoral Fellow supported by a grant from the United States Children's Bureau (Project No. 417). This work was supported in part by Dr. Henry C. Buswell and Bertha H. Buswell Research Fellowship funds, State University of New York at Buffalo, and by United States Public Health Service Grant GM-I5874 from the National Institute of General Medical Sciences. We wish to thank Drs. Eric Barnard and Mario C. Rattazzi for helpful advice and reviewing the manuscript and Mrs. A. Clark and Mrs. A Wright for technical assistance. REFERENCES C. A. ZITTLE, E. S. DELLAMONICA AND J. H. CUSTER, Arch. Biochem. Biophys., 48 (1953) 43. M. G. P. J. WARRINGA AND J. A. COHEN, Biochim. Biophys. Acta, 16 (1955) 3 oo. F. BERGMANN AND R. SEGAL, Biochim. Biophys. dcta, 16 (1955) 513 . M. H. COLEMAN AND D. D. ELEY, Bioehim. Biophys. Acta, 67 (1963) 646. G. L. ELLMAN, K. D. COURTNEY, at-. ANDRES AND R. M. FEATHERSTONE, Biochem. Pharmacol., 7 (1961) 88. 6 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J . Biol. Chem., 193 (1951) 265. 7 J. T. DOD6E, C. MITCHELL AND D. J. HANAHAN, Arch. Biochem. Biophys., IOO (1963) 119. 8 G. K. ACKERS, Biochemistry, 3 (1964) 723 . 9 M. J. I~ARNOVSKY AND L. ROOTS, J. Histochem. Cytochem., 12 (1964) 219. i o C. 1. WRIGHT AND J. C. SABRINE, J. Pharmaeol. Exptl. Therap., 93 (1948) 230. [1 J. CHANGEUX, A. RYTER, W. LEUZINGER, P. BARRAND AND T. PODLESKI, Proe. Natl. Acad. •c. U.S., 62 (1969) 986. 12 W. LEUZINGER, M. GOLDBERG AND E. CAUVIN, J. ~'~lo1. Biol., 4 ° (1969) 217. 13 M. B. BELLHORN, (). O. BLUMENFELD AND P. M. GALLOP, Biochem. Biophys. Res. Commun., 39 (197o) 267. 14 R. L. JACKSON AND M. H. APRISON, dr. Neurochem., 13 (1966) 1351. 15 I. }(. H o AND G. L. ELLMAN, dr. Neurochem., 16 (1969) 15o 5. I 2 3 4 5

Bioehim. Biophys. Acta, 236 (1971) 612-618