STUDIES ON CHOLINESTERASE*

STUDIES ON CHOLINESTERASE* III. PURIFICATION OF THE ENZYME FROM ELECTRIC TISSUE BY FRACTIONAL AMMONIUM SULFATE PRECIPITATION BY MORTIMER A. ROTHENBERG AND DAVID NACHMANSOHN (From the Departments of Neurology and Biochemistry, College of Physicians and Surgeons, Columbia University, New York) (Received for publication, January 27,1947) It has been shown that nerve and muscle tissues contain an esterase easily distinguished from all other esterases by its high relative specificity for acetylcholine (10). This enzyme, cholinesterase, is present in all types of nerves and muscles throughout the animal kingdom, and has been found in the heads of Tubularia, a hydrozoan coelenterate, the lowest animal form to possess neuromuscular tissue comparable to that of higher animals (5). Substances which are inhibitors of the enzyme also abolish conduction in nerve and muscle (2). This abolition is reversible when the enzyme inhibition is also reversible, whereas irreversible inhibition of the enzyme is paralleled by irreversible inhibition of conduction. A striking parallelism has been obtained between the behavior of the chemical and electrical processes if their irreversibility is tested as a function of time and temperature (3, 4, 6). In connection with a great number of other facts, the evidence appears to be conclusive that the activity of cholinesterase is inseparably associated with the conduction of impulses along nerve and muscle fibers (7, 8). It is obvious that an enzyme of such apparent importance in the functioning of nerve and muscle is of great interest and that more information is desirable concerning its chemical and physicochemical properties. It is useful to obtain the enzyme in purified form in order to facilitate detailed studies concerning its chemical and physicochemical properties. In this paper will be described a procedure by which cholinesterase of a high degree of purity has been obtained by fractional ammonium sulfate precipitation of extracts from the electric organ of Electrophorus electricus. The same procedure always yields nearly identical results. Studies of the physicochemical properties of the enzyme will be reported in subsequent papers. Electric tissue has been selected because it splits per hour amounts of acetylcholine equivalent to 1 to 3 times its own weight. This extraordinarily high concentration of the enzyme is particularly interesting in view of the low protein (2 per cent) and high water content (92 per cent). It is rare to find a material which offers itself so favorably as a source for * This work was made possible by a grant of the Josiah Macy, Jr., Foundation. 223

224 CHOLINESTERASE. III enzyme purification. It was found, in 1938, that cholinesterase may be easily extracted from these organs and obtained in cell-free solution by homogenization and centrifugation (9). Certain properties of this enzyme have been described. It has also been shown that the esterase in these organs is exclusively specific cholinesterase (10). Methods The manometric method was used for the determination of the enzyme activity in the usual way (10). The buffer solution in the vessel contained 0.13 i~ NaCl, 0.04 M MgC&, and 0.025 in NaHCO, in final concentration. The substrate was placed in the side bulb in 0.1 cc., its final concentration after mixing being 0.018 M. If the enzyme solution was diluted to a very high degree, gelatin was added (0.1 per cent final concentration), since it was found that the enzyme in high dilution becomes unstable. The protein content was determined by drying and weighing. 1 cc. of the enzyme solution was put into a small centrifuge tube of 3 cc. capacity and of known weight (between 4.0 and 4.5 gm.). 1 cc. of 20 per cent trichloroacet,ic acid was added. The precipitate was left to settle overnight. The tube was centrifuged at 4000 R.P.M. for about 10 minutes and the supernatant was discarded. The precipitate was washed twice with 2 cc. of distilled water. After the supernatant of the second washing had been removed, the tube was kept in a drying oven at 110 and weighed on the following day. The dialysis of the purified cholinesterase solutions was carried out in the following manner: The enzyme, after solution in secondary phosphate plus phosphate buffer, was poured into a bag made of cellophane casing, a small glass bead was added, and the open end then tied. Allowance was made for a sufficiently large air bubble to cause stirring of the solution. The cellophane bag was then attached to an eccentric stirrer and immersed in a 3 liter jar. The stirrer was rotated by means of a small gear reduction motor. For the 1st day dialysis was carried out against 0.1 M Na*HPOJ. The outer dialysis fluid was changed every l$ hours. In all six to seven changes of secondary phosphate (3 liters each) were used. The outer dialysis fluid was changed to 0.1 M NaCl, 0.015 M MgC12,0.015 M phosphate buffer, ph 7.4, in the final change. During the 2nd and 3rd days, the dialysis was continued against the latter solution, changes being made every 2 hours. In all, six to seven changes were made each day. Results Preparation of Material-The electric organ contains an appreciable amount of mucin (1)) which proved to be most disturbing in earlier attempts

M. A. ROTHENBERG AND D. NACHMANSOHN 225 at purification of cholinesterase extracted from this tissue. It could, however, be easily removed in the following manner: the tissue was cut into small pieces and allowed to remain in the refrigerator under toluene for 1 month. Each day there appears some exudate which has a high mucin content. The exudate is discarded periodically and finally, at the end of 4 to 5 weeks, the tissue contains very little mucin. In the preparation to be described here, 10.05 kilos of electric tissue from nine electric eels (Electrophorus electricus) were treated in the way mentioned above. At the end of 1 month, 6.44 liters of exudate had been removed. The remaining 3.6 kilos of tissue were ground in a Waring blendor with 9.2 liters of 5 per cent ammonium sulfate. During the first grinding, 250 cc. of ammonium sulfate were added to each 100 gm. portion TABLE Cholinesterase Activities and Protein Contents of Solutions Obtained during Purijication of Enzyme from Electric Tissue of Electrophorus electricus The last column indicates the enzyme activity per unit of protein (AP = mg. of acetylcholine split per hour per mg. of protein). Preparation 8 has been obtained by high speed centrifugation of a solution similar to Preparation 7. Preparation No Dilution used 1 1:300 2 1:1,200 3 1:5,000 4 1:10,000 5 1:10,000 6 1:10,000 7 1:20,000 8 1:200,000 co1 output ~.nwn. $er hr. 392 360 301 220 368 442 303 323 I ACh split Protein *mm. $er cc. per hr. mg. @2r cc. 0.950 3.3 3.5 8.0 12.2 4.3 17.8 4.9 29.8 6.1 35.8 4.8 49.2 2.3 524.0 7.0 AP 288 438 2,810 3,630 4,890 7,460 21,400 75,000 of tissue. The second extraction was carried out with two-thirds the volume of the first extraction. In this way, 10.24 liters of solution were obtained. The enzyme activity and the protein content of this solution and of those obtained during the course of purification are recorded in Table I. 1 cc. of this solution was able to split 0.95 gm. of acetylcholine per hour (Table I, Preparation 1). The total amount of solution prepared was then able to split 9.7 kilos of acetylcholine per hour. 1 mg. of protein of this solution could split 288 mg. of acetylcholine per hour. The activity of the preparation per unit of protein will be called A,. In this instance A, was consequently 288. PuriJication of Cholinesterase Solution First Step of Purification-Solid ammonium sulfate was added to the 10.24 liters of solution in sufficient quantity to make the concentration of

226 CHOLINES'I'ERASE. III ammonium sulfate 15 per cent. The precipitate formed was centrifuged and discarded, since it was known that at a concentration of 15 per cent ammonium sulfate only negligible amounts of cholinesterase are precipitated. To the supernatant solution was added solid ammonium sulfate to increase the concentration to 40 per cent. The precipitate was filtered by suction. The filter paper was comminut,ed and suspended in 1350 cc. of 5 per cent ammonium sulfate. The ph was adjusted to 7.1. The paper was filtered off through gauze and washed first in 750 cc. of 5 per cent ammonium sulfate (ph 7.0) and then a second time in 400 cc. of 5 per cent ammonium sulfate. Each of these washings was then combined with the original solution. 2.54 liters of solution were obtained in this way. 1 cc. was able to split 3.5 gm. of acetylcholine per hour. The activity of the whole amount was 8.9 kilos. The AP was 438 (Table I, Preparation 2). The solution was centrifuged at 2000 R.P.M. for 13 minutes to remove the mucin-like substance floating in the solution. The enzyme activity was unaffected by this operation. About one-half of this solution, 1.25 liters, was precipitated by the addition of sufficient saturated ammonium sulfate to bring the concentration up to 19 per cent. At this concentration of ammonium sulfate some loss of cholinesterase occurs, but, at the same time, much of the protein is removed and a considerable degree of purification is obtained. The ph was adjusted to 5.82. The solution was left in the refrigerator overnight in order to allow the precipitate to digest and settle. The precipitate was centrifuged at 2000 R.P.M. in an anglehead centrifuge and washed first with 450 cc. and then with 300 cc. of 19 per cent ammonium sulfate (ph 5.82). The washings were combined. The supernatant fluid and washings were precipitated separately at 29 per cent ammonium sulfate by the addition of saturated ammonium sulfate (ph 5.62). Most of the enzyme is precipitated at this concentration of ammonium sulfate at the slightly acid ph used. The solution was then centrifuged at 2000 R.P.M. The supernatant was discarded. Three-fourths of the protein was dissolved in 113 cc. of 5 per cent ammonium sulfate (ph was adjusted to 7.3). The remaining fourth of the total protein was dissolved in 25 cc. of 0.1 M NaCl, 0.015 M MgCl,, 0.015 M phosphate buffer, ph 7.4, plus 5 cc. of 0.1 M Na2HP04. This solution was dialyzed for 3 days. At the end of the dialysis 52 cc. of solution were obtained. 1 cc. of this solution was able to split 12.2 gm. of acetylcholine per hour. The activity of the whole amount was 634 gm. of acetylcholine per hour. The AP was 2810 (Table I, Preparation 3). The remaining 1160 cc. of solution with an AP of 438 were precipitated

M. A. ROTHENRERQ AND D. NACHMANSOHN 227 successively at 19 and 29 per cent ammonium sulfate and treated in a manner similar to the previous fraction. The precipitate obtained at 29 per cent was dissolved in 120 cc. of 5 per cent ammonium sulfate and combined with the corresponding solution of the first fraction in which three-fourths of the protein had been dissolved in 113 cc. of 5 per cent ammonium sulfate. The volume of the two solutions combined was 300 cc., 1 cc. of which was capable of splitting 17.8 gm. of acetylcholine per hour. The activity of the whole volume was 5.3 kilos. AP was 3630 (Table I, Preparation 4). Second Step of PuriJication-This solution was precipitated with 19 per cent ammonium sulfate (ph 5.83). The precipitate was allowed to settle for 2 hours, was centrifuged at 2000 R.P.M., and the precipitate discarded. To the supernatant was added saturated ammonium sulfate to 28 per cent (ph 5.57). The precipitate was centrifuged on the next day and dissolved in 100 cc. of 5 per cent ammonium sulfate (ph 7.0). 148 cc. of a solution were obtained and were able to split 29.8 gm. of acetylcholine per hour per cc. The activity of the whole volume was 4.41 kilos. A, was 4890 (Table I, Preparation 5). 40 cc. of this solution were precipitated at 28 per cent with saturated ammonium sulfate (ph 5.66). The precipitate was allowed to remain in the refrigerator overnight to settle and then centrifuged at 5000 R.P.M. for 15 minutes. The precipitate was dissolved in 6 cc. of 0.1 M NaCl, 0.015 M MgCh, 0.015 M phosphate buffer, ph 7.4, plus 6 cc. of 0.1 M Na2HP04. 17 cc. of this solution were dialyzed for 3 days. 19 cc. of a solution were obtained. 1 cc. of this solution was able to split 35.8 gm. of acetylcholine per hour. The activity of the whole volume was consequently 680 gm. AP was 7460 (Table I, Preparation 6). Third Step of PuriJication-105 cc. of solution with AP 4890 were precipi,tated at 20.5 per cent ammonium sulfate and centrifuged at 5000 R.P.M. after 3 hours. The supernatant was saved and the precipitate was washed three times with 30 cc. of 20.5 per cent ammonium sulfate (ph 6.2). The supernatant and washings (kept separated from the supernatant) were precipitated at 27 per cent with saturated ammonium sulfate. The precipitate was centrifuged at 3500 R.P.M. in the refrigerated centrifuge at 10. The combined precipitates were dissolved in 35 cc. of 1 per cent ammonium sulfate. The above solution was then precipitated at 27 per cent with saturated ammonium sulfate (ph 5.61) and allowed to remain in the refrigerator for 3 hours. This was centrifuged in the cold centrifuge as above, and the precipitate dissolved in 7.5 cc. of 0.1 M NaCl, 0.015 M MgC&, 0.015 M phosphate buffer, ph 7.4, plus 7.5 cc. of 0.1 M NtiHPO,. The solution

228 CHOLINESTERASE. III was dialyzed for 3 days. This and the other dialyzed solutions were used for ultracentrifuge studies, which will be described elsewhere. 28.5 cc. of solution were obtained. 1 cc. was able to split 49.2 gm. of acetylcholine per hour. The activity of the whole amount was 1.40 kilos. A, was 21,400 (Table I, Preparation 7). Speci$city Pattern cf Puri$ed Cholinesterase-If the rates of hydrolysis of different esters by a solution of cholinesterase purified from electric tissue are tested, it is found that the same pattern is obtained as with the freshly prepared suspension (10). The highest A, at which the patt,ern was previously tested was 3000. It appeared of interest to see whether or not a still higher degree of purification affects the pattern observed earlier. In Table II the data obtained with preparations of an increasing degree of TABLE Specijicity of Cholinesterase from Electric Tissue of Electrophorus electricus, Tested by Rate of Hydrolysis of Digerent Esters, in Course of Purification QZ = the hydrolysis rate of Substrate z in per cent of that of acetylcholine. Pr = propionylcholine, Bu = butyrylcholine, Me = acetyl-p-methylcholine (mecholyl). q of benzoylcholinr tributyrine, and methylbutyrate was always 0. AP 270 1,365 2,500 75,000 ACh split c.mm. per hr. 335 311 320 584 qpr 97 101 109 108 purity are summarized. The solution with an A, of 21,000 was an enzyme solution prepared by fractional ammonium sulfate precipitation, as described above. If an enzyme solution purified in this way is centrifuged at high speed, the enzyme is found in the pellet. One of the solutions obtained from a pellet suspension after ultracentrifugation at 48,000 R.P.M. was capable of splitting 525 gm. of acetylcholine per cc. per hour and had a protein content of only G to 7 mg. per cc., the A, thus being at least 75,000. Since this solution appeared to have only one component in an analytical ultracentrifuge run, it seems quite probable that this is virtually the pure enzyme and that the pattern found is consequently that of the pure cholinesterase of the material used. As can be seen from Table II, all the solutions tested have a pattern virtually identical with that of the homogenized suspension. Consequently, the pattern found is that of the pure cholinesterase of the material used. II de 22 31 18 21

M. A. ROTHENBERG AND D. NACHMANSOHN 229 DISCUSSION The results described show that a high degree of purity of cholinesterase may be easily obtained by fractional ammonium sulfate precipitation once the mucin present in the electric tissue has been removed. A great part of the protein which is not cholinesterase protein may be precipitated by the ammonium sulfate up to a concentration of 21 per cent, whereas most of the enzyme protein remains in solution. Thereafter, ammonium sulfate at concentrations up to 27 per cent precipitates practically all of the cholinesterase protein, leaving the other proteins in solution. If the original solution obtained after the removal of the mucin is precipitated with ammonium sulfate at 21 per cent, a great part of cholinesterase is precipitated simultaneously and would be lost. Also, if the original solution is precipitated by ammonium sulfate at a concentration of 27 per cent, many other proteins would be precipitated simultaneously and the degree of purity obtained would be much lower. Moreover, part of the enzyme remains in solution and would be lost. The purification has therefore been carried out in several steps in order to avoid the large losses incurred by immediate precipitation at these concentrations. The precipitation with 40 per cent of ammonium sulfate and filtration of the precipitate in the first step were applied only after it had been repeatedly found that centrifugation of the precipitate at this or slightly lower concentrations of ammonium sulfate was very difficult with the centrifuge available. It is possible that with a more efficient centrifuge than that used it would be feasible to separate the precipitate from the supernatant fluid. It is also important to observe the slight acidification described (ph 5.6 to 5.9). At this ph precipitations are more complete. Stronger acidification, however, should be avoided, since the enzyme is apparently sensitive to greater ph changes. In a freshly prepared homogenized suspension of electric tissue of Electrophorus electricus, 1 mg. of protein splits about 50 to 100 mg. of acetylcholine per hour (A, = 50 to 100). Since the A, rises up to more than 20,000 by fractional ammonium sulfate precipitation, a 200- to 400-fold purification has been obtained. An AP of 20,000 means that in spite of the extraordinarily high concentration of cholinesterase in electric tissue and in spite of its low protein content, the enzyme forms less than 0.5 per cent of the tissue protein. But even this figure is much too high. By high speed centrifugation, the enzyme protein may be further separated from other proteins, indicating that the enzyme protein forms less than 1 per thousand of the total. Since the amount of enzyme protein is so small in the electric organ,

230 CHOLIN ESTERASE. III the high speed of acetylcholine hydrolysis suggests the possibility of a great efficiency of the enzyme system. Experiments with high speed centrifugation carried out in collaboration with Dr. Kurt G. Stern, which will be described in a separate paper, indicate that the molecular weight of the enzyme is in the neighborhood of 3 million and that its turnover number is approximately 20 million per minute. Thus, 1 enzyme molecule seems to be able to split 1 molecule of acetylcholine in 3 to 4 millionths of a second. If these results &an be confirmed by the investigations still in progress, they would be noteworthy not only from a purely biochemical view. In the face of the assumption that the release and removal of acetylcholine are associated directly with the electrical manifestations of the surface membrane in nerve and muscle, the high potential rate of acetylcholine inactivation is of considerable physiological interest. SUMMARY A method is described by which a highly purified solution of cholinesterase is obtained from the electric organ of Electrophorus electricus. The mucin present in the organ is removed and fractional ammonium sulfate precipitation is used in carrying out the separations. In a concentration of ammonium sulfate up to 21 per cent, the enzyme protein remains in solution, whereas many other proteins are precipitated and may thus be removed. By precipitation with ammonium sulfate at a concentration of 27 per cent, the cholinesterase is nearly completely precipitated, whereas other proteins remain in solution at that concentration. In this way, an enzyme solution may be obtained in which 1 mg. of protein splits 20,000 to 21,000 mg. of acetylcholine per hour. Since in a freshly homogenized suspension of the tissue used 1 mg. of protein splits 50 to 100 mg. of acetylcholine, the preparation obtained represents a 200- to 400-fold purification. The precipitations with ammonium sulfate have to be carried out in several steps. A further separation of inactive protein from the enzyme protein may be obtained by high speed centrifugation. The pattern obtained by testing the rate of hydrolysis of different esters is typical for cholinesterase and remains unchanged throughout the whole process of purification. It is still the same if a higher degree of purification is obtained by high speed centrifugation. Thus, the esterase present in electric tissue seems to be exclusively cholinesterase. BIBLIOGRAPFIY 1. Bailey, K., Biochem. J., 33, 255 (1939). 2. Bullock, T. H., Nachmansohn, D., and Rothenberg, M. A., J. Neurophysiol., 9, 9 (1946).

M. A. ROTHENBERG AND D. NACHbfANSOHN 231 3. Bullock, T. H., Grundfest, H., Nachmansohn, D., Rothenberg, M. A., and Sterling, K., J. Neurophysiol., 9, 253 (1946). 4. Bullock, T. H., Grundfest, H., Nachmansohn, D., and Rothenberg, M. A., J. Neurophysiol., lo,63 (1947). 5. Bullock, T. H., Grundfest, H., Nachmansohn, D., and Rothenberg, M. A., J. Neurophysiol., 10, 11 (1947). 6. Grundfest, H., Nachmansohn, D., and Rothenberg, M. A., J. Neurophysiol., in press. 7. Nachmansohn, D., in Harris, R. S., and Thimann, K. V., Vitamins and hormones, New York, 3,337 (1945). S:Nachmansohn, D., Ann. New York Ad. SC., 47,395 (1946). 9. Nachmansohn, D., and Lederer, E., Bull. sot. chim. biol., 21, 797 (1939). 10. Nachmansohn, D., and Rothenberg, M. A., J. BioZ. Chem., 168, 653 (1945).

STUDIES ON CHOLINESTERASE: III. PURIFICATION OF THE ENZYME FROM ELECTRIC TISSUE BY FRACTIONAL AMMONIUM SULFATE PRECIPITATION Mortimer A. Rothenberg and David Nachmansohn J. Biol. Chem. 1947, 168:223-231. Access the most updated version of this article at http://www.jbc.org/content/168/1/223.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/168/1/223.citation.full.h tml#ref-list-1