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Substrate specificity studies on bee acetylcholinesterase purified by gradient centrifugation
J. Ins. Physiol., 1963, t-01. 9, pp. 495 to 507. Pergaon
Press Ltd.
Printed in Great Britain
SUBSTRATE SPECIFICITY STUDIES ON BEE ACETYLCHOLINESTERASE PURIFIED BY GRADIENT CENTRIFUGATION RALPH E. KUNKEE and GUNTER ZWEIG Agricultural Toxicology
and Residue Research Laboratory, Davis, California, U.S.A. (Remitted 25 February
University of California,
1963)
Abstract-Acetylcholinesterase was purified from bees by ammonium sulphate fractionation and sucrose gradient centrifugation. The final preparation had an overall enrichment of SO-fold, specific activity based on protein, or 200-fold, specific activity based on total organic material. The specific activity of the best preparation was 12 units &mole substrate hydrolysed/min)/mg protein. The purified preparation was classified as true acetylcholinesterase on the basis of substrate specikity. The final purification seemed to be free of pseudocholinesteraae and other esterases. The hydrolysis of acetyl-,%methylcholine by the purified preparation indicated that the enzyme from bees is different from acetylcholinesterase from most other sources. The pattern of centrifugation of the enzyme in sucrose gradient was not greatly changed by treatment with high salt, urea, mercaptoethanol, desoxycholate, butanol, or sonic oscillation. The purified preparation was activated by low levels of butanol. INTRODUCTION ACETYLCHOLINESTEFME, an important enzyme in the function of nervous tissue, has been studied in many organisms. Insect acetylcholinesterase has received special attention following the discovery that the inhibition of the enzyme is the probable mechanism of activity of many insecticidal materials, e.g. the organophosphates and carbamates. In spite of this great interest, nearly all of the investigations with insect acetylcholinesterases have been carried out on crude homogenates. Purification procedures from only two insect sources have appeared: LORD (1961) obtained a twelve-fold purification (specific activity based on nondialysable, 280 my. absorbing material) of acetylcholinesterase from the German cockroach and DAUTERMAN et al. (1962) purified the enzyme 157-fold (based on total organic material) from the house fly. These preparations can be compared to one of the most active acetylcholinesterase preparations, the 200- to 400-fold purification obtained from the electric eel (ROTHENBERG and NACHMANSOHN, 1947; LAWLER, 1959). Thus, there remains a need for additional purified preparations from insects, and especially from different insect species. In contrast to the properties of the enzyme from most other sources (AUGUSTINSSON, 1949), METCALFet al. (1955)
495
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RALPH
E. KUNKEFI AND GUNTER Zwmc
found that acetylcholinesterase activity in crude homogenates of some insect species, especially the honey bee and the American cockroach, was not inhibited by high concentrations of the substrate, acetyl+?-methylcholine. WOLFE and SMALLMAN (1956) also found that the crude enzyme from bees was not inhibited by acetyl-/3methylcholine, except at extremely high concentrations (1.0 M). The lack of substrate inhibition in crude homogenates might be the result of activity of more than one enzyme (METCALFand MARCH,1955), or that the enzyme in bees might be different from acetylcholinesterase from other sources. Other results from studies with cholinesterase inhibitors have also indicated that bee enzyme might be different from other acetylcholinesterases, at least different from that found in the house fly (METCALFand MARCH,1950). Substrate inhibition studies with a purified enzyme preparation from bees would help establish whether or not bee acetylcholinesterase is different from other acetylcholinesterases. The partial purification of acetylcholinesterase from bees is described in this paper. Ammonium sulphate fractionation and sucrose gradient centrifugation were used for the purification. Gradient centrifugation has been used for the purification of large molecular weight material such as viruses (BRAKXE,1953) and was found to be well suited for use in purification of acetylcholinesterase. Studies on substrate inhibition of the purified material support the hypothesis that the bee enzyme is, indeed, different from acetylcholinesterases from other sources. METHODS Enzyme assays Acetylcholinesterase activity was measured for the purification steps by the method of ELLMAN et al. (1961), involving the calorimetric determination of sulfhydryl groups produced by enzymic hydrolysis of acetylthiocholine. To test-tubes containing 3.2 to 3.5 ml of O-1 M sodium phosphate, pH 8.0, and O-2 ml of colour reagent [OGO63 M S,S’-dithiobis2-nitrobenzoic acid (Aldrich Chemical Co.) in 0.065 M Na phosphate, pH 7.0, containing 1 mg/ml NaHCOs] was added, at timed intervals, 0.2 ml substrate [OGO95 M acetylthiocholine iodide (Calif. Corp. for Biochemical Research), prepared bi-weekly and stored in the cold] and O-1 to 0.3 ml of material to be assayed. The tubes were incubated for timed intervals, 15-30 min. in a water bath at 30°C. The absorbancy of the colour formed was measured at the end of the incubation time in a Spectronic 20 Colorimeter at 412 mp. Reagent blanks and controls containing 10ms M eserine (Mann Research Laboratories) were also incubated. Cholinesterases are inhibited by lo+ M eserine (WILSON, 1960) and the activity resulting from non-specific esterases was determined by measuring enzyme activity in the presence of eserine. The reaction was found to be linear with time and enzyme concentration to an absorbancy of 1 (O-382 mole thiocholine produced under these conditions) and all determinations reported were in this range. Units of activity were taken as net pmoles of thiocholine produced per min. Addition of Mg’+ did not increase the activity of the enzyme preparation at any stage in the purification. A titrimetric assay was employed for substrate specificity studies. Enzymic hydrolysis was followed by automatic titration of the liberated acetic acid with the use of Metrohm Impulsomate E373 pH-Stat. Various amounts of material to be assayed were added to 0.15 ml of Michel’s II buffer, containing sodium barbital, sodium phosphate, and sodium chloride (MICEIEL, 1949), 0.3 ml of 0.9% (w/v) NaCl solution, 0.15 ml substrate, and H,O to give a final volume of 3.0 ml. One-thousandth N NaOH was automatically added to maintain pH 7.8. The reaction was carried out at 30°C. Non-cholinesterase hydrolysis of
STUDIES ON BEE ACETYLCHOLINESTERASE
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the substrate was determined by measuring the hydrolysis after the addition of 0.1 ml of 10m3 M eserine. Units of activity were taken as net pmoles of NaOH added per min.
The material to be assayed was precipitated in 0.3 M trichloroacetic acid and washed with this reagent. The protein was then determined by the biuret method of GORNALL et al. (1949). Bovine serum albumin (Nutritional Biochem. Corp.) was used as a standard.
Total organic material The method of JOHNSON (1949),as modified by DAUTRRMAN et al. (1962),was used to measure total organic material. Bovine serum albumin and ghxose were used as standards.
Sucrose gradient tubes were prepared by successive layering of 1.4 ml, 2-8 ml, 2.8 ml, 2.8 ml, and I.4 ml of 25, 20, 15, 10, and 5% (w/v) sucrose, respectively, in cellulose centrifuge tubes for the No. 40 rotor of the Spinco Model L Ultracentrifuge. Each sucrose solution was made up to have a final concentration of 9.0% (w/v) NaCI. After the tubes were stored one day in the cold, @8 ml of the enzyme preparation was floated on top of the solution and the tubes were then centrifuged at 105,000 g for the times indicated in the text. Fractions of 0.8 mI were collected through a pinhole pierced in the bottom of the tube, and like fractions were combined.
Substrates Besides acetylthiocholine iodide, the following substrates were used: acetylcholinebromide (Eastman Organic Chemicals), DL-acetyl-B-methylcholine chloride (Mann Research Laboratories), butyrylcholine iodide (Mann Research Laboratories), butyrylthiochoiine iodide (Calif. Corp. for Biochemical Research), and benzoylcholine chloride(Calif. Corp. for Biochemical Research). RESULTS
Preparation
ofltomogeszate
Worker honey bees, A$& mellifera L., of American-Italian stock, were collected under mild smoke anaesthesia and stored at - 20°C until used. The heads were removed from the bodies by shaking the frozen bees with dry ice (MOOREFIELD, 1957). The heads were then isolated by successive sieving lirst through a 6- then an 8-in. mesh wire screen. Batches of 300 to 500 heads were ground in a cold mortar and pestle with 0.05 ml H*O/head for 2 min. Several batches of brei were combined and strained through 2 layers of cheese cloth. The residue was re-ground for 2 min with 0.01 ml H,O/head and re-strained. The brei had an activity of about 0.2 units/head, based on the calorimetric assay. In our first experiments. we stored the brei overnight, at room temperature, and the activity was often higher after this treatment (about 15 per cent) (MIXCALF and MARCH, 1949). Higher activity also could be obtained by preparing the homogenate with a hand-driven Potter-Elvehjem glass homogenizer. However, after the solubilization treatment (see below) the e~e-~~~~head was comparable regardless of method of grinding or overnight incubation. The homogenate was then lyophiliied and stored at 4°C. 33
498
RALPH
E.KUNKEBAND
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Solubilization of en2yme activity The acetylcholinesterase of the homogenate was found to be particulate, since 85 per cent of the activity sedimented after 15 min centrifugation at 40,000 g. We found no greater degree of solubilization if the pH of the homogenate was varied from pH 6.5 to 8.5 (SMALLMANand WOLFE, 1956). Certain particulate enzymes have been solubiliied by treatment with organic solvents (MORTON, 1950). Dry butanol treatment of lyophilized material has been used to solubilize acetylcholinesterase from red blood cells and fly heads (DAUTERMAN et al., 1962; COHENand WARRINGA, 1953). This procedure also was found satisfactory for solubilixing of the bee enzyme. Treatment of the wet brei, however, with butanol or toluene was not satisfactory. The enzyme was solubilized as follows: the lyophilized homogenate was stirred in the cold with 1-butanol (0.3 ml/head) which had been dried over Na$O,. After 1 hr, the preparation was centrifuged (10 min at 10,000 g) and the residual butanol was removed from the combined pellets by vacuum. The resulting material was re-suspended in HsO (0.02 ml/head) with 10 pg/ml ribonuclease (Nutritional Biochemicals Corp.) and 5 pg/ml deoxyribonuclease (Nutritional Biochemicals Corp.), and stored at room temperature for 1.5 hr, and then centrifuged 15 min at 20,000 g. About half the original acetylcholinesterase activity was found in the dark red, clear, supematant fluid. Attempts to obtain a greater degree of solubilization by varying the amount of butanol and the duration of the butanol treatment were not successful. The nuclease treatment also had no effect on the degree of solubilixation. An attempt was made to remove the bulk of the soluble protein, before solubilization of the enzyme, by centrifugation. However, butanol treatment of the lyophilized sediment of the homogenate hardly brought about any solubilixation of the acetylcholinesterase. Purification of the bee acetylcholinesterase after solubilization by chromatography on modified cellulose was attempted, but the results were unsatisfactory. Very little recovery or purification was obtained by using diethylaminoethyl-, ‘ECTEOLA-‘, aminoethyl-, or carboxymethyl-cellulose. Ammonium @hate
fractionation
Purification of the enzyme by ammonium sulphate precipitation was carried out in the presence of pH 8.0 buffer. The specific activity of the precipitate formed increased with increasing ammonium sulphate concentration from about 18 to 23% (w/v). Fractionation in the absence of buffer or at pH 4-O brought about little purification (cf. LAWLER,1959). The ammonium sulphate fractionation was performed, in the cold, as follows: to a measured volume of solubilized, nuclease-treated, supematant fluid (see above) were added 2.4 volumes of O-1 M sodium phosphate, pH 8.0, and, dropwise, with stirring, 1.8 volumes of 5076 ammonium sulphate. After being stored overnight, the material was centrifuged for 10 min at 10,000 g. To the supernatant fluid was added 1.1 volume of ammonium sulphate solution as before. After about 5 hr, the the material was centrifuged again, and the precipitate was suspended in a minimal
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volume of 1 M NaCl~-~5 M Na phosphate, pH 7.0. The ammonium sulphate preparation was then dialysed for a total of 4.5 hr against 1 1. of dialysis medium (1 M NaCl-OGE M Na phosphate, pH 7-O), which was changed twice. During dialysis, the dark brown suspension went into solution. After dialysis, the ratio of absorbancies at 280 rnp and 260 rnp was unity.
Acetylcholinesterase from the electric eel has been shown to be large, apparently l-10 million molecular weight (LAWLER, 1961). If the bee enzyme is of comparable size, differential centrifugation could be used as a purification step. Fig. 1 shows the activity remaining in the supematant after centrifugation at 105,000 g (Spine0 Model L, No. 40 rotor) for various lengths of time. Although the enzyme was easily sedimented under these conditions, the pellet was a gummy material which we were unable to re-suspend. Sucrose gradient centrifugation, however, proved to be successful for purification.
FIG. 1. Acetylcholinesterase activity remaining in supematant fluid after centrifugation at 105,000 g for the indicated times.
In a preliminary experiment, a sucrose gradient of 60-10x (wlv) sucrose in isotonic NaCl was used. After 2 hr centrifugation, the peak of the enzyme activity was separated from the peak of the protein (the latter measured by absorbancy at 280 mp). Subsequently, better separation of the enzyme from the bulk of the protein was obtained by centrifugation for 6 hr in a sucrose gradient of 25 to 5%
RALPH
500
E.
KIJNKEE AND GUNTER
ZWEIG
(w/v) sucrose in 9*00/o (w/v) NaCl. A typical purification is shown in Fig. 2. The fractions with the higher specific activity were pale yellow to light brown in colour, and nearer the top of the tube, in which the specific activity was considerably lower, the colour was increasingly pink. The fractions with the highest specific activity, numbers 3 to 7 in Fig. 2, were combined and dialysed for a total of 4.5 hr, against three changes of media, to remove salt and sugar. The first and second dialysis media were 3 1. of 0.0154 M NaC1-0~005 M Na phosphate, pH 7.0, and the third was 0.0154 M NaCl. After centrifugation and dialysis, the enzyme activity was
r 1)1~
J-
0
fraction FIG. sulphate in 9.0% y!:(b) .
2. Sucrose gradient centrifugation of acetylcholinesterase after ammonium fractionation. Centrifugation was carried out in 25 to 5% sucrose gradient NaCl for 6 hr at 105,000g. ( a) acetylcholinesterase activity (units/ml) protein absorbency at 280 rnp (0) ; and (c) specific activity (units/A&
dilute and was stable for only a few days. Concentration of the material by perevaporation or ultrafiltration resulted in a large loss of enzyme activity. Usually the material was concentrated by lyophilization, which resulted in a 10 to 30 per cent loss of activity. A second gradient centrifugation increased the purity of the material slightly. Fig. 3 shows the results of a re-centrifugation in a sucrose gradient in isotonic NaCl for 2 hr. (The lyophilized sample was dissolved in a small volume of H,O and dialysed against isotonic NaCl before the re-centrifugation.) The correlation of the
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activity peak with one of the protein peaks indicated this was the best purification that could be obtained by this procedure. In both the first and second centrifugation, the band of acetylcholinesterase activity was rather wide. Gradient centrifugation in a swinging bucket head (Spinco rotor No. SW39L) for 3 hr at 125,OOOg resulted in almost identical results to those given above using the angle head. Other attempts to narrow the band of activity were made by centrifugation in presence of reagents which would tend to dissociate any aggregation of the enzyme or any association of the enzyme with
-
I
Ol
1
5
fraction
IO
I
15
FIG. 3. Second gradient centrifugation of acetylcholineaterase. Centrifugation was carried out in 25 to 5% sucrose in @9% NaCl for 2 hr at 105,000 g. (a) acetylch~lineaterast activity (units/ml) (0); (b) protein (absorbency at 280 rnp) (0); and (c) specific activity (units/A,~) (---).
large non-active particles. In addition to the centrifugation in high salt (9.0% NaCl, see above), gradient centrifugation was carried out in pH 8.0 buffer, urea, mercaptoethanol, I-butanol, and desoxycholate. A sample subjected to sonic In no case was the band of enzyme oscillation (10 KC) was also centrifuged. activity any narrower, and the peak of the enzyme activity sedimented closer to the bulk of the rest of the protein, as compared to the centrifugation in the absence of these treatments.
86.5 s-5* 1.6*
4.50 4.98 6.6
7.6 2.2
1.47
1.0
Step enrichment
49.5t
1.0 2.0 4.6 33.8
0.24 0.47 1.09 8.10
11*9t
Overall enrichment
Units mg
Based on protein
* About 1%of the lyophilized preparation was used for the second centrifugation. t Calculated from step enrichment based on 280 rnp absorbency.
2nd centrifugation
Lyophilized
1140 710 309 97.8
Brei Solubilized Ammonium sulphate 1st centrifugation
100 62 27 8.6
Units/A,s,,
Yield (%I
Units
Purification stage
Enzyme activity
Based on 280my. absorbency
TABLE ~-PURIFICATION OF ACETYLCHOLINESTERASE
11.9t
8.10
0.057
Units mg
208t
142
1.0
Overall enrichment
Based on total organic material
61.5 5.7 1.5 -
Eserine resistant activity Units
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of ac~t~lc~~n~st~ase
Table 1 gives a protocol of a typical purification of the enzyme, starting with 5000 bee heads. The overall enrichment is given in terms of both protein and total organic material, in order to compare it with purification data given by other workers (see Introduction). Also listed in Table 1 is the activity determined in the presence of 10d M eserine. About 5-10 per cent of the total esterase activity of the crude brei was insensitive to eserine and represented non-cholinesterase activity (WILSON, 1960). In the purified preparation, no non-cholinesterase activity was found, as determined by the calorimetric or titrimetric assay.
The purified preparation was characterized by its esterase activity against several substrates. In Fig. 4, the enzymic hydrolysis, using the calorimetric assay, at various concentrations of acetylthiocholine and butyrylthiocholine, is shown.
6
5
4
PS
3
2
FIG. 4. Substrate-activity curves for acetylcholinesterase. The calorimetric assay, based on thiocholine produced, was used. Equal concentrations of activity were used for each curve in Figs. 4 and 5. (a) purified enzyme and acetylthiocholine (ATCh) (O-O); (b) brei and ATCh (O---O); (c) purified enzyme and butyryithiocholine (BuTCh) (A-A); and (d) brei and BuTCh (A---&. pS is the negative logarithm of the substrate concentration.
For comparison, the activity of the crude brei is also presented. The concentration of purified enzyme and brei assayed was adjusted to give an activity of 0.1 units, with the calorimetric assay, under optimal conditions. The results with acetylthiocholine show the typical substrate inhibition curve with both the purified was much slower. preparation and the brei. The hydrolysis of bu~~l~ocholine The purified preparation also showed substrate inhibition with this substrate, but the crude brei shows little substrate inhibition up to a butyrylthiocholine concentration of 0.01 M. Above 0.01 M the non-enzymic hydrolysis at pH 8 of both of
504
RALPH E. KUNKEEANDGUNTERZWEIC
these substrates is too great to make the calorimetric assay practical (ELL~+~AN et al., 1961). The titrimetric assay was employed for the measurement of activity of the The enzymic hydrolysis of acetylcholine, enzyme against other substrates. butyrylcholine, benzoylcholine, and acetyl-/3-methylcholine is given in Fig. 5. The concentration of enzyme used for each determination was the same as that used for the calorimetric results in Fig. 4. Again, the natural substrate acetylcholine shows substrate inhibition, as do the methyl-analog and butyrylcholine. Substrate inhibition with butyrylcholine and the slow hydrolysis of benzoylcholine indicate the lack of pseudocholinesterase (AUCXJSTINSSON, 1949; HEATH, 1961) in the purified preparation. Also shown in Fig. 5 is the butyrylcholinesterase activity of the crude
4-5 40
$0
ps
2.0
I.0
0.0
FIG. 5. Substrate-activity curves for acetylcholinesterase. The titrimetric assay, based on acetic acid produced, was used. Equal concentrations of activity were used for each curve in Figs. 4 and 5. Curves a, b, c, d are with the purified enzyme, and e, with the brei. (a) acetylcholine (ACh) (0-O); (b) acetyl-@methylcholine (AMeCh) (O-O); (c) butyrylcholine (BuCh) (x-x ); (d) benzoylcholine (B&h) (A-A); and (e) BuCh (O--- 9). pS is the negative logarithm of the substrate concentration.
brei. Substrate inhibition is not evident in this case, and the results confirm the presence of pseudocholinesterase in the crude brei (METCALFet al., 1955) which is removed during the purification. It should be noted that in contrast to the
STUDIBS ON BEE ACBTYLCHOLINBS-fEBASE PURIFIED BY GRADIENT CENTRIFWGATION
505
purified bee enzyme, the enzyme purified from flies by DAUTERMAN et al. (1962) hydrolyses butyrylcholine rather rapidly. The results with a~e~l-~-m~yl~ho~ne, showing substrate inhibition with the purified preparation, but with an optimal concent~tion several orders of magnitude greater than the natural substrate, require special comment and will be considered in the Discussion. Activation with butanol Bee acetylcholinesterase was found to be more active in the presence of low concentrations of butanol and was similar in this respect to acetylcholinesterase in the American cockroach thoracic ganglia ~~OLHO~, 1961) and house fly heads (DAet al., 1962). In the presence of O-03 ml l-butanol in the calorimetric assay, the activity of the purified preparation was increased l&fold. These results with the purified preparation agree with the suggestion of COLHOUN (1961) that the effect of the solvent is on the active sites of the enzyme and not the elimination of some inhibitor or an increase in enzyme concentration. DISCUSSION By use of the following criteria LION, 1960; HEATH,1961), we have classified the enzyme preparation obtained from bees as acetylcholinesterase : 1. The enzyme is inhibited by 10d M eaerine. 2. The enzyme shows substrate inhibition with acetylcholine and butyrykholine. 3. The enzyme hydrolyses acetylcholine at a faster rate than butyrylcholine. 4. The enzyme hydrolyses acetyl-@nethylcholine rapidly and benzoylcholine slowly. The tirat criterion classties the enzyme as a cholinesterase, while the other criteria define the enzyme as true acetylcholinesteraae rather than pseudocholineaterase. The optimal substrate concentration for hydrolysis of acetylcholine by the purified preparation from bees was found to be about 3-8 x 10d M (see Fig. 5). This is one-tenth the optimal concentration of acetylcholine usually given in the literature (AUGUSTINSSON, 1949; METCALFand MARCH,1950; HEATH,1961). This discrepancy may be explained in that, in the latter instances, the determinations had been made at pH’s nearer 7. Our measurements were made at pH 8 (optimal) and confirm the study by WOLFEand SMALLMAN (1956) who showed that the optimal substrate ~neen~tion was pH dependent. The hydrolysis of acetyl-fl-methylcholine by the purified enzyme preparation is unusual. Not only is the maximal activity nearly twice that found with acetylcholine, but the optimal substrate concentration with acetyl-p-methylcholine is over 300 times that of acetylcholine (or 150 times, if only one optical isomer is considered). In contrast, the typical hydrolysis pattern of acetyl-fi-methylcholine by true acetylcholinesterase is an optimal concentration near that of acetylcholine but at a slower rate (AU~USTIN~ON, 1949; METCALFet al., 1955). Furthermore, NEWALF et al. (1955) showed no substrate inhibition with Abel-~-rne~yl~ho~e with the crude bee homogenate, but they did not test at concentrations of substrate as high as those tested here. However, their results may show a levelling off of
506
RALPH
E. KUNK~E ANDGIJNTERZWEIC
activity at a concentration not far above that which shows maximal activity in our experiments. As these workers have suggested, a lack of substrate inhibition in the crude brei hydrolysis of the methyl-analog of acetylcholine may be explained by the presence of another enxyme in the brei which hydrolyses this substrate at high concentrations. The high optimal substrate concentration with acetyl-fi-methylcholine with the purified bee enzyme supports the suggestion presented above, that the bee enzyme is different from other true acetylcholinesterases. Enzymes from three other species tested by &&TCAl.F et al. (1%5), viz. P~~l~~a a??mtkm, and A&eta astid, are possibly also in the Pcpmmymex barbatyes caiifd, category with the bee enzyme. The difference between acetylcholinesterase from bees and that from other sources, with respect to optimal concentration and maximal activity with the substrate acetyl+methylcholine, is significant because it may reflect differences in the enzyme surface near the active sites. The results can be represented as >K~w&Kucw where K,,uBwI,) GBfAMeoN ‘&&4oht and Guw#&A.M~) are the dissociation (Michaelis-Menten) constants of the enxymeand &.4Ch, substrate complexes for acetylcholine and acetyl-fi-methylcholine, respectively, and K’(WCW and ~(ACW are the corresponding dissociation constants for the second molecule of the respective substrate (HEATH,1961). In other words, the association of the bee enzyme with anal-~-m~yl~oline may be inhibited by the steric hindrance between the p-methyl group on the substrate and a part of the enzyme surface which is unique to the bee enxyrne. Furthermore, the shape of the surface of the bee enzyme is such that the steric hindrance would be even more important in the association of the enxyme-substrate complex with a second molecule of substrate, at either the anionic site or the esteratic site, and thus the second association is even more greatly inhibited with the bee enzyme. Further examination of this suggestion could reveal molecular structures which would not be inhibitory to the bee enzyme, but which would inhibit other acetylcholinesterases. Although the total enrichment of the enzyme is somewhat better than that for et al., 1962), the specific activity other insect preparations (LOW, 1961; DAUTERMAN of the finalpreparation from bees is not especiallyhigh. The bee preparation with the highest specific activity was about 12 units/mg protein, and can be compared to 27 units/mg total organic material (at 37°C) found for the purified preparation from house Aies (DAUTERMAN et al., 1962). These activities are comparable to the specific activity of the starting material used in the electric eel preparation, 8-3 unitsimg protein (LAWLXR,1959). The low specific activity of the insect preparations may reflect the degree of impurity of the starting material, as compared to the electric eel extract, or to low activity of the enzyme, per se. Acknowledgements-This work was supported by a grant from the United States Public Health Service (No. EF-183). (A preliminary report was presented at the Pacific Slope Biochemical Conference in Seattle, Washington (U.S.A.), September 1962.1 We are grateful to Mr. LEE WATKINSof the University Apiary for obtaining the bees, to the Department of Avian Medicine for the use of the Spinco Centrifuge, and to the Department of Biochemistry for the use of the Metrohm pH-Stat.
STUDIESON BEE ACETYl..CHOLINESI’ERASE PURIFIEDBY GRADIENT CENTRIFUGATION
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REFERENCES AUGUSTINSSONK.-B. (1949) Substrate concentration and specificity of choline estersplitting enzymes. Arch. Biochem. 23, 11 l-126. BRAKKEM. K. (1953) Zonal separations by density-gradient eentrifu~tion. Ar&. 3ioeh~. Biophys. 45, 275-290. COHEN J. A. and WARRINGAM. G. P. J. (1953) Purification of cholinesterase from ox red cells. Biochem. biophys. Actu 10, 195-196. COLHOUN E. H. (1961) Activation of cockroach acetylcholinesterase by water-miscible organic solvents. Nature, Lond. 189, 309-310. DAUTEFZMAN W. C., TALENS A., and VAN A~PERENK. (1962) Partial purification and properties of fiyhead cholinesterase. J. Ins. Physiol. 8, l-14. ELLMAN G. L., COURTNEYK. D., ANDRES V., and FEATHERSTO~JE R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity, Bioc&. Phannacol. 7, 88-95. GORNALL A. G., BARDAWXLLC. J., and DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction, J, biol. Chem. 177, 751 -766. HEATH D. F. (1961) O~gunophosphorus Poisons, pp. 103-115. Pergamon Press, New York. JOHNSONM. J. (1949) A rapid micromethod for estimation of non-volatile organic matter. J. biol. Chem. 181, 707-711. LADLER H. C. (1959) A simplified procedure for the partial purification of acetylcholinesterase from electric tissue. J. bioi. Chem. 234, 799-801. LADLER H. C. (1961) Structural aspects of a~~icho~ester~. Fed. P~oc. 20, A-381f. Lozn K. A. (1961) The partial purification and properties of a cholinesterase from Bkzttdla germanica L. Bi0them.J. 78,4834%. METCALFR. L. and MARCHR. B. (1949) Studies on the mode of action of parathion and its derivatives and their toxicity to insects. J. econ. Ent. 42, 721-728. METCALF R. L. and MARCH R. B. (1950) Properties of acetylcholinester from the bee, the fly, and the mouse; and their relation to insecticide action. J. econ. Ent. 43.670-677. METCALF R. L., MARCH R. B., and MAXON M. G. (1955) Substrate properties of insect cholinesterasea. Amr. ent. Sac. Amer. 48, 222-228. MICHEL H. 0. (1949) An electrometric method for the determination of red blood cell and plasma chohnesterase activity. J. Lab. c&n. Med. 34,1564-1568. MOOREFIELDH. H. (1957) Improved method of harvesting house fly heads for use in cholinesterase studies. Contr. Boyce Thompson Inst. 18, 463. MORTON R. K. (1950) Separation and purification of enzymes associated with insoluble particles. Nature, Lomb. 166, 1092-1095. ROTHENBERGM. A. and NACHMANSOHN D. (1947) Studies on cholinesterase.-III. Purification of the enzyme from electric tissue by fractional ammonium sulfate precipitation. J. biol. Chem. 168, 223-231. SMALLMAN B. N. and WOLFE L. S. (1956) Soluble and particulate cholinesterase in insects. y. cell. camp. Fhysiol~ 48, 197-213. WILSON I. B. (1960) Acetylchohnesterase. In The Enzymes (Ed. by BOYER P. D., LAY H., and MYRBXCKK.), Vol. 4, 501-520. Academic Press, New York. WOLFE L. S. and SMALLMAN B. N. (1956) The properties of cholinesterases from insects. J. cell. camp. Physiol. 48, 215-235.
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SUBSTRATE SPECIFICITY STUDIES ON BEE ACETYLCHOLINESTERASE PURIFIED BY GRADIENT CENTRIFUGATION RALPH E. KUNKEE and GUNTER ZWEIG Agricultural Toxicology
and Residue Research Laboratory, Davis, California, U.S.A. (Remitted 25 February
University of California,
1963)
Abstract-Acetylcholinesterase was purified from bees by ammonium sulphate fractionation and sucrose gradient centrifugation. The final preparation had an overall enrichment of SO-fold, specific activity based on protein, or 200-fold, specific activity based on total organic material. The specific activity of the best preparation was 12 units &mole substrate hydrolysed/min)/mg protein. The purified preparation was classified as true acetylcholinesterase on the basis of substrate specikity. The final purification seemed to be free of pseudocholinesteraae and other esterases. The hydrolysis of acetyl-,%methylcholine by the purified preparation indicated that the enzyme from bees is different from acetylcholinesterase from most other sources. The pattern of centrifugation of the enzyme in sucrose gradient was not greatly changed by treatment with high salt, urea, mercaptoethanol, desoxycholate, butanol, or sonic oscillation. The purified preparation was activated by low levels of butanol. INTRODUCTION ACETYLCHOLINESTEFME, an important enzyme in the function of nervous tissue, has been studied in many organisms. Insect acetylcholinesterase has received special attention following the discovery that the inhibition of the enzyme is the probable mechanism of activity of many insecticidal materials, e.g. the organophosphates and carbamates. In spite of this great interest, nearly all of the investigations with insect acetylcholinesterases have been carried out on crude homogenates. Purification procedures from only two insect sources have appeared: LORD (1961) obtained a twelve-fold purification (specific activity based on nondialysable, 280 my. absorbing material) of acetylcholinesterase from the German cockroach and DAUTERMAN et al. (1962) purified the enzyme 157-fold (based on total organic material) from the house fly. These preparations can be compared to one of the most active acetylcholinesterase preparations, the 200- to 400-fold purification obtained from the electric eel (ROTHENBERG and NACHMANSOHN, 1947; LAWLER, 1959). Thus, there remains a need for additional purified preparations from insects, and especially from different insect species. In contrast to the properties of the enzyme from most other sources (AUGUSTINSSON, 1949), METCALFet al. (1955)
495
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RALPH
E. KUNKEFI AND GUNTER Zwmc
found that acetylcholinesterase activity in crude homogenates of some insect species, especially the honey bee and the American cockroach, was not inhibited by high concentrations of the substrate, acetyl+?-methylcholine. WOLFE and SMALLMAN (1956) also found that the crude enzyme from bees was not inhibited by acetyl-/3methylcholine, except at extremely high concentrations (1.0 M). The lack of substrate inhibition in crude homogenates might be the result of activity of more than one enzyme (METCALFand MARCH,1955), or that the enzyme in bees might be different from acetylcholinesterase from other sources. Other results from studies with cholinesterase inhibitors have also indicated that bee enzyme might be different from other acetylcholinesterases, at least different from that found in the house fly (METCALFand MARCH,1950). Substrate inhibition studies with a purified enzyme preparation from bees would help establish whether or not bee acetylcholinesterase is different from other acetylcholinesterases. The partial purification of acetylcholinesterase from bees is described in this paper. Ammonium sulphate fractionation and sucrose gradient centrifugation were used for the purification. Gradient centrifugation has been used for the purification of large molecular weight material such as viruses (BRAKXE,1953) and was found to be well suited for use in purification of acetylcholinesterase. Studies on substrate inhibition of the purified material support the hypothesis that the bee enzyme is, indeed, different from acetylcholinesterases from other sources. METHODS Enzyme assays Acetylcholinesterase activity was measured for the purification steps by the method of ELLMAN et al. (1961), involving the calorimetric determination of sulfhydryl groups produced by enzymic hydrolysis of acetylthiocholine. To test-tubes containing 3.2 to 3.5 ml of O-1 M sodium phosphate, pH 8.0, and O-2 ml of colour reagent [OGO63 M S,S’-dithiobis2-nitrobenzoic acid (Aldrich Chemical Co.) in 0.065 M Na phosphate, pH 7.0, containing 1 mg/ml NaHCOs] was added, at timed intervals, 0.2 ml substrate [OGO95 M acetylthiocholine iodide (Calif. Corp. for Biochemical Research), prepared bi-weekly and stored in the cold] and O-1 to 0.3 ml of material to be assayed. The tubes were incubated for timed intervals, 15-30 min. in a water bath at 30°C. The absorbancy of the colour formed was measured at the end of the incubation time in a Spectronic 20 Colorimeter at 412 mp. Reagent blanks and controls containing 10ms M eserine (Mann Research Laboratories) were also incubated. Cholinesterases are inhibited by lo+ M eserine (WILSON, 1960) and the activity resulting from non-specific esterases was determined by measuring enzyme activity in the presence of eserine. The reaction was found to be linear with time and enzyme concentration to an absorbancy of 1 (O-382 mole thiocholine produced under these conditions) and all determinations reported were in this range. Units of activity were taken as net pmoles of thiocholine produced per min. Addition of Mg’+ did not increase the activity of the enzyme preparation at any stage in the purification. A titrimetric assay was employed for substrate specificity studies. Enzymic hydrolysis was followed by automatic titration of the liberated acetic acid with the use of Metrohm Impulsomate E373 pH-Stat. Various amounts of material to be assayed were added to 0.15 ml of Michel’s II buffer, containing sodium barbital, sodium phosphate, and sodium chloride (MICEIEL, 1949), 0.3 ml of 0.9% (w/v) NaCl solution, 0.15 ml substrate, and H,O to give a final volume of 3.0 ml. One-thousandth N NaOH was automatically added to maintain pH 7.8. The reaction was carried out at 30°C. Non-cholinesterase hydrolysis of
STUDIES ON BEE ACETYLCHOLINESTERASE
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the substrate was determined by measuring the hydrolysis after the addition of 0.1 ml of 10m3 M eserine. Units of activity were taken as net pmoles of NaOH added per min.
The material to be assayed was precipitated in 0.3 M trichloroacetic acid and washed with this reagent. The protein was then determined by the biuret method of GORNALL et al. (1949). Bovine serum albumin (Nutritional Biochem. Corp.) was used as a standard.
Total organic material The method of JOHNSON (1949),as modified by DAUTRRMAN et al. (1962),was used to measure total organic material. Bovine serum albumin and ghxose were used as standards.
Sucrose gradient tubes were prepared by successive layering of 1.4 ml, 2-8 ml, 2.8 ml, 2.8 ml, and I.4 ml of 25, 20, 15, 10, and 5% (w/v) sucrose, respectively, in cellulose centrifuge tubes for the No. 40 rotor of the Spinco Model L Ultracentrifuge. Each sucrose solution was made up to have a final concentration of 9.0% (w/v) NaCI. After the tubes were stored one day in the cold, @8 ml of the enzyme preparation was floated on top of the solution and the tubes were then centrifuged at 105,000 g for the times indicated in the text. Fractions of 0.8 mI were collected through a pinhole pierced in the bottom of the tube, and like fractions were combined.
Substrates Besides acetylthiocholine iodide, the following substrates were used: acetylcholinebromide (Eastman Organic Chemicals), DL-acetyl-B-methylcholine chloride (Mann Research Laboratories), butyrylcholine iodide (Mann Research Laboratories), butyrylthiochoiine iodide (Calif. Corp. for Biochemical Research), and benzoylcholine chloride(Calif. Corp. for Biochemical Research). RESULTS
Preparation
ofltomogeszate
Worker honey bees, A$& mellifera L., of American-Italian stock, were collected under mild smoke anaesthesia and stored at - 20°C until used. The heads were removed from the bodies by shaking the frozen bees with dry ice (MOOREFIELD, 1957). The heads were then isolated by successive sieving lirst through a 6- then an 8-in. mesh wire screen. Batches of 300 to 500 heads were ground in a cold mortar and pestle with 0.05 ml H*O/head for 2 min. Several batches of brei were combined and strained through 2 layers of cheese cloth. The residue was re-ground for 2 min with 0.01 ml H,O/head and re-strained. The brei had an activity of about 0.2 units/head, based on the calorimetric assay. In our first experiments. we stored the brei overnight, at room temperature, and the activity was often higher after this treatment (about 15 per cent) (MIXCALF and MARCH, 1949). Higher activity also could be obtained by preparing the homogenate with a hand-driven Potter-Elvehjem glass homogenizer. However, after the solubilization treatment (see below) the e~e-~~~~head was comparable regardless of method of grinding or overnight incubation. The homogenate was then lyophiliied and stored at 4°C. 33
498
RALPH
E.KUNKEBAND
GUNNERZWEIC
Solubilization of en2yme activity The acetylcholinesterase of the homogenate was found to be particulate, since 85 per cent of the activity sedimented after 15 min centrifugation at 40,000 g. We found no greater degree of solubilization if the pH of the homogenate was varied from pH 6.5 to 8.5 (SMALLMANand WOLFE, 1956). Certain particulate enzymes have been solubiliied by treatment with organic solvents (MORTON, 1950). Dry butanol treatment of lyophilized material has been used to solubilize acetylcholinesterase from red blood cells and fly heads (DAUTERMAN et al., 1962; COHENand WARRINGA, 1953). This procedure also was found satisfactory for solubilixing of the bee enzyme. Treatment of the wet brei, however, with butanol or toluene was not satisfactory. The enzyme was solubilized as follows: the lyophilized homogenate was stirred in the cold with 1-butanol (0.3 ml/head) which had been dried over Na$O,. After 1 hr, the preparation was centrifuged (10 min at 10,000 g) and the residual butanol was removed from the combined pellets by vacuum. The resulting material was re-suspended in HsO (0.02 ml/head) with 10 pg/ml ribonuclease (Nutritional Biochemicals Corp.) and 5 pg/ml deoxyribonuclease (Nutritional Biochemicals Corp.), and stored at room temperature for 1.5 hr, and then centrifuged 15 min at 20,000 g. About half the original acetylcholinesterase activity was found in the dark red, clear, supematant fluid. Attempts to obtain a greater degree of solubilization by varying the amount of butanol and the duration of the butanol treatment were not successful. The nuclease treatment also had no effect on the degree of solubilixation. An attempt was made to remove the bulk of the soluble protein, before solubilization of the enzyme, by centrifugation. However, butanol treatment of the lyophilized sediment of the homogenate hardly brought about any solubilixation of the acetylcholinesterase. Purification of the bee acetylcholinesterase after solubilization by chromatography on modified cellulose was attempted, but the results were unsatisfactory. Very little recovery or purification was obtained by using diethylaminoethyl-, ‘ECTEOLA-‘, aminoethyl-, or carboxymethyl-cellulose. Ammonium @hate
fractionation
Purification of the enzyme by ammonium sulphate precipitation was carried out in the presence of pH 8.0 buffer. The specific activity of the precipitate formed increased with increasing ammonium sulphate concentration from about 18 to 23% (w/v). Fractionation in the absence of buffer or at pH 4-O brought about little purification (cf. LAWLER,1959). The ammonium sulphate fractionation was performed, in the cold, as follows: to a measured volume of solubilized, nuclease-treated, supematant fluid (see above) were added 2.4 volumes of O-1 M sodium phosphate, pH 8.0, and, dropwise, with stirring, 1.8 volumes of 5076 ammonium sulphate. After being stored overnight, the material was centrifuged for 10 min at 10,000 g. To the supernatant fluid was added 1.1 volume of ammonium sulphate solution as before. After about 5 hr, the the material was centrifuged again, and the precipitate was suspended in a minimal
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volume of 1 M NaCl~-~5 M Na phosphate, pH 7.0. The ammonium sulphate preparation was then dialysed for a total of 4.5 hr against 1 1. of dialysis medium (1 M NaCl-OGE M Na phosphate, pH 7-O), which was changed twice. During dialysis, the dark brown suspension went into solution. After dialysis, the ratio of absorbancies at 280 rnp and 260 rnp was unity.
Acetylcholinesterase from the electric eel has been shown to be large, apparently l-10 million molecular weight (LAWLER, 1961). If the bee enzyme is of comparable size, differential centrifugation could be used as a purification step. Fig. 1 shows the activity remaining in the supematant after centrifugation at 105,000 g (Spine0 Model L, No. 40 rotor) for various lengths of time. Although the enzyme was easily sedimented under these conditions, the pellet was a gummy material which we were unable to re-suspend. Sucrose gradient centrifugation, however, proved to be successful for purification.
FIG. 1. Acetylcholinesterase activity remaining in supematant fluid after centrifugation at 105,000 g for the indicated times.
In a preliminary experiment, a sucrose gradient of 60-10x (wlv) sucrose in isotonic NaCl was used. After 2 hr centrifugation, the peak of the enzyme activity was separated from the peak of the protein (the latter measured by absorbancy at 280 mp). Subsequently, better separation of the enzyme from the bulk of the protein was obtained by centrifugation for 6 hr in a sucrose gradient of 25 to 5%
RALPH
500
E.
KIJNKEE AND GUNTER
ZWEIG
(w/v) sucrose in 9*00/o (w/v) NaCl. A typical purification is shown in Fig. 2. The fractions with the higher specific activity were pale yellow to light brown in colour, and nearer the top of the tube, in which the specific activity was considerably lower, the colour was increasingly pink. The fractions with the highest specific activity, numbers 3 to 7 in Fig. 2, were combined and dialysed for a total of 4.5 hr, against three changes of media, to remove salt and sugar. The first and second dialysis media were 3 1. of 0.0154 M NaC1-0~005 M Na phosphate, pH 7.0, and the third was 0.0154 M NaCl. After centrifugation and dialysis, the enzyme activity was
r 1)1~
J-
0
fraction FIG. sulphate in 9.0% y!:(b) .
2. Sucrose gradient centrifugation of acetylcholinesterase after ammonium fractionation. Centrifugation was carried out in 25 to 5% sucrose gradient NaCl for 6 hr at 105,000g. ( a) acetylcholinesterase activity (units/ml) protein absorbency at 280 rnp (0) ; and (c) specific activity (units/A&
dilute and was stable for only a few days. Concentration of the material by perevaporation or ultrafiltration resulted in a large loss of enzyme activity. Usually the material was concentrated by lyophilization, which resulted in a 10 to 30 per cent loss of activity. A second gradient centrifugation increased the purity of the material slightly. Fig. 3 shows the results of a re-centrifugation in a sucrose gradient in isotonic NaCl for 2 hr. (The lyophilized sample was dissolved in a small volume of H,O and dialysed against isotonic NaCl before the re-centrifugation.) The correlation of the
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activity peak with one of the protein peaks indicated this was the best purification that could be obtained by this procedure. In both the first and second centrifugation, the band of acetylcholinesterase activity was rather wide. Gradient centrifugation in a swinging bucket head (Spinco rotor No. SW39L) for 3 hr at 125,OOOg resulted in almost identical results to those given above using the angle head. Other attempts to narrow the band of activity were made by centrifugation in presence of reagents which would tend to dissociate any aggregation of the enzyme or any association of the enzyme with
-
I
Ol
1
5
fraction
IO
I
15
FIG. 3. Second gradient centrifugation of acetylcholineaterase. Centrifugation was carried out in 25 to 5% sucrose in @9% NaCl for 2 hr at 105,000 g. (a) acetylch~lineaterast activity (units/ml) (0); (b) protein (absorbency at 280 rnp) (0); and (c) specific activity (units/A,~) (---).
large non-active particles. In addition to the centrifugation in high salt (9.0% NaCl, see above), gradient centrifugation was carried out in pH 8.0 buffer, urea, mercaptoethanol, I-butanol, and desoxycholate. A sample subjected to sonic In no case was the band of enzyme oscillation (10 KC) was also centrifuged. activity any narrower, and the peak of the enzyme activity sedimented closer to the bulk of the rest of the protein, as compared to the centrifugation in the absence of these treatments.
86.5 s-5* 1.6*
4.50 4.98 6.6
7.6 2.2
1.47
1.0
Step enrichment
49.5t
1.0 2.0 4.6 33.8
0.24 0.47 1.09 8.10
11*9t
Overall enrichment
Units mg
Based on protein
* About 1%of the lyophilized preparation was used for the second centrifugation. t Calculated from step enrichment based on 280 rnp absorbency.
2nd centrifugation
Lyophilized
1140 710 309 97.8
Brei Solubilized Ammonium sulphate 1st centrifugation
100 62 27 8.6
Units/A,s,,
Yield (%I
Units
Purification stage
Enzyme activity
Based on 280my. absorbency
TABLE ~-PURIFICATION OF ACETYLCHOLINESTERASE
11.9t
8.10
0.057
Units mg
208t
142
1.0
Overall enrichment
Based on total organic material
61.5 5.7 1.5 -
Eserine resistant activity Units
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of ac~t~lc~~n~st~ase
Table 1 gives a protocol of a typical purification of the enzyme, starting with 5000 bee heads. The overall enrichment is given in terms of both protein and total organic material, in order to compare it with purification data given by other workers (see Introduction). Also listed in Table 1 is the activity determined in the presence of 10d M eserine. About 5-10 per cent of the total esterase activity of the crude brei was insensitive to eserine and represented non-cholinesterase activity (WILSON, 1960). In the purified preparation, no non-cholinesterase activity was found, as determined by the calorimetric or titrimetric assay.
The purified preparation was characterized by its esterase activity against several substrates. In Fig. 4, the enzymic hydrolysis, using the calorimetric assay, at various concentrations of acetylthiocholine and butyrylthiocholine, is shown.
6
5
4
PS
3
2
FIG. 4. Substrate-activity curves for acetylcholinesterase. The calorimetric assay, based on thiocholine produced, was used. Equal concentrations of activity were used for each curve in Figs. 4 and 5. (a) purified enzyme and acetylthiocholine (ATCh) (O-O); (b) brei and ATCh (O---O); (c) purified enzyme and butyryithiocholine (BuTCh) (A-A); and (d) brei and BuTCh (A---&. pS is the negative logarithm of the substrate concentration.
For comparison, the activity of the crude brei is also presented. The concentration of purified enzyme and brei assayed was adjusted to give an activity of 0.1 units, with the calorimetric assay, under optimal conditions. The results with acetylthiocholine show the typical substrate inhibition curve with both the purified was much slower. preparation and the brei. The hydrolysis of bu~~l~ocholine The purified preparation also showed substrate inhibition with this substrate, but the crude brei shows little substrate inhibition up to a butyrylthiocholine concentration of 0.01 M. Above 0.01 M the non-enzymic hydrolysis at pH 8 of both of
504
RALPH E. KUNKEEANDGUNTERZWEIC
these substrates is too great to make the calorimetric assay practical (ELL~+~AN et al., 1961). The titrimetric assay was employed for the measurement of activity of the The enzymic hydrolysis of acetylcholine, enzyme against other substrates. butyrylcholine, benzoylcholine, and acetyl-/3-methylcholine is given in Fig. 5. The concentration of enzyme used for each determination was the same as that used for the calorimetric results in Fig. 4. Again, the natural substrate acetylcholine shows substrate inhibition, as do the methyl-analog and butyrylcholine. Substrate inhibition with butyrylcholine and the slow hydrolysis of benzoylcholine indicate the lack of pseudocholinesterase (AUCXJSTINSSON, 1949; HEATH, 1961) in the purified preparation. Also shown in Fig. 5 is the butyrylcholinesterase activity of the crude
4-5 40
$0
ps
2.0
I.0
0.0
FIG. 5. Substrate-activity curves for acetylcholinesterase. The titrimetric assay, based on acetic acid produced, was used. Equal concentrations of activity were used for each curve in Figs. 4 and 5. Curves a, b, c, d are with the purified enzyme, and e, with the brei. (a) acetylcholine (ACh) (0-O); (b) acetyl-@methylcholine (AMeCh) (O-O); (c) butyrylcholine (BuCh) (x-x ); (d) benzoylcholine (B&h) (A-A); and (e) BuCh (O--- 9). pS is the negative logarithm of the substrate concentration.
brei. Substrate inhibition is not evident in this case, and the results confirm the presence of pseudocholinesterase in the crude brei (METCALFet al., 1955) which is removed during the purification. It should be noted that in contrast to the
STUDIBS ON BEE ACBTYLCHOLINBS-fEBASE PURIFIED BY GRADIENT CENTRIFWGATION
505
purified bee enzyme, the enzyme purified from flies by DAUTERMAN et al. (1962) hydrolyses butyrylcholine rather rapidly. The results with a~e~l-~-m~yl~ho~ne, showing substrate inhibition with the purified preparation, but with an optimal concent~tion several orders of magnitude greater than the natural substrate, require special comment and will be considered in the Discussion. Activation with butanol Bee acetylcholinesterase was found to be more active in the presence of low concentrations of butanol and was similar in this respect to acetylcholinesterase in the American cockroach thoracic ganglia ~~OLHO~, 1961) and house fly heads (DAet al., 1962). In the presence of O-03 ml l-butanol in the calorimetric assay, the activity of the purified preparation was increased l&fold. These results with the purified preparation agree with the suggestion of COLHOUN (1961) that the effect of the solvent is on the active sites of the enzyme and not the elimination of some inhibitor or an increase in enzyme concentration. DISCUSSION By use of the following criteria LION, 1960; HEATH,1961), we have classified the enzyme preparation obtained from bees as acetylcholinesterase : 1. The enzyme is inhibited by 10d M eaerine. 2. The enzyme shows substrate inhibition with acetylcholine and butyrykholine. 3. The enzyme hydrolyses acetylcholine at a faster rate than butyrylcholine. 4. The enzyme hydrolyses acetyl-@nethylcholine rapidly and benzoylcholine slowly. The tirat criterion classties the enzyme as a cholinesterase, while the other criteria define the enzyme as true acetylcholinesteraae rather than pseudocholineaterase. The optimal substrate concentration for hydrolysis of acetylcholine by the purified preparation from bees was found to be about 3-8 x 10d M (see Fig. 5). This is one-tenth the optimal concentration of acetylcholine usually given in the literature (AUGUSTINSSON, 1949; METCALFand MARCH,1950; HEATH,1961). This discrepancy may be explained in that, in the latter instances, the determinations had been made at pH’s nearer 7. Our measurements were made at pH 8 (optimal) and confirm the study by WOLFEand SMALLMAN (1956) who showed that the optimal substrate ~neen~tion was pH dependent. The hydrolysis of acetyl-fl-methylcholine by the purified enzyme preparation is unusual. Not only is the maximal activity nearly twice that found with acetylcholine, but the optimal substrate concentration with acetyl-p-methylcholine is over 300 times that of acetylcholine (or 150 times, if only one optical isomer is considered). In contrast, the typical hydrolysis pattern of acetyl-fi-methylcholine by true acetylcholinesterase is an optimal concentration near that of acetylcholine but at a slower rate (AU~USTIN~ON, 1949; METCALFet al., 1955). Furthermore, NEWALF et al. (1955) showed no substrate inhibition with Abel-~-rne~yl~ho~e with the crude bee homogenate, but they did not test at concentrations of substrate as high as those tested here. However, their results may show a levelling off of
506
RALPH
E. KUNK~E ANDGIJNTERZWEIC
activity at a concentration not far above that which shows maximal activity in our experiments. As these workers have suggested, a lack of substrate inhibition in the crude brei hydrolysis of the methyl-analog of acetylcholine may be explained by the presence of another enxyme in the brei which hydrolyses this substrate at high concentrations. The high optimal substrate concentration with acetyl-fi-methylcholine with the purified bee enzyme supports the suggestion presented above, that the bee enzyme is different from other true acetylcholinesterases. Enzymes from three other species tested by &&TCAl.F et al. (1%5), viz. P~~l~~a a??mtkm, and A&eta astid, are possibly also in the Pcpmmymex barbatyes caiifd, category with the bee enzyme. The difference between acetylcholinesterase from bees and that from other sources, with respect to optimal concentration and maximal activity with the substrate acetyl+methylcholine, is significant because it may reflect differences in the enzyme surface near the active sites. The results can be represented as >K~w&Kucw where K,,uBwI,) GBfAMeoN ‘&&4oht and Guw#&A.M~) are the dissociation (Michaelis-Menten) constants of the enxymeand &.4Ch, substrate complexes for acetylcholine and acetyl-fi-methylcholine, respectively, and K’(WCW and ~(ACW are the corresponding dissociation constants for the second molecule of the respective substrate (HEATH,1961). In other words, the association of the bee enzyme with anal-~-m~yl~oline may be inhibited by the steric hindrance between the p-methyl group on the substrate and a part of the enzyme surface which is unique to the bee enxyrne. Furthermore, the shape of the surface of the bee enzyme is such that the steric hindrance would be even more important in the association of the enxyme-substrate complex with a second molecule of substrate, at either the anionic site or the esteratic site, and thus the second association is even more greatly inhibited with the bee enzyme. Further examination of this suggestion could reveal molecular structures which would not be inhibitory to the bee enzyme, but which would inhibit other acetylcholinesterases. Although the total enrichment of the enzyme is somewhat better than that for et al., 1962), the specific activity other insect preparations (LOW, 1961; DAUTERMAN of the finalpreparation from bees is not especiallyhigh. The bee preparation with the highest specific activity was about 12 units/mg protein, and can be compared to 27 units/mg total organic material (at 37°C) found for the purified preparation from house Aies (DAUTERMAN et al., 1962). These activities are comparable to the specific activity of the starting material used in the electric eel preparation, 8-3 unitsimg protein (LAWLXR,1959). The low specific activity of the insect preparations may reflect the degree of impurity of the starting material, as compared to the electric eel extract, or to low activity of the enzyme, per se. Acknowledgements-This work was supported by a grant from the United States Public Health Service (No. EF-183). (A preliminary report was presented at the Pacific Slope Biochemical Conference in Seattle, Washington (U.S.A.), September 1962.1 We are grateful to Mr. LEE WATKINSof the University Apiary for obtaining the bees, to the Department of Avian Medicine for the use of the Spinco Centrifuge, and to the Department of Biochemistry for the use of the Metrohm pH-Stat.
STUDIESON BEE ACETYl..CHOLINESI’ERASE PURIFIEDBY GRADIENT CENTRIFUGATION
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REFERENCES AUGUSTINSSONK.-B. (1949) Substrate concentration and specificity of choline estersplitting enzymes. Arch. Biochem. 23, 11 l-126. BRAKKEM. K. (1953) Zonal separations by density-gradient eentrifu~tion. Ar&. 3ioeh~. Biophys. 45, 275-290. COHEN J. A. and WARRINGAM. G. P. J. (1953) Purification of cholinesterase from ox red cells. Biochem. biophys. Actu 10, 195-196. COLHOUN E. H. (1961) Activation of cockroach acetylcholinesterase by water-miscible organic solvents. Nature, Lond. 189, 309-310. DAUTEFZMAN W. C., TALENS A., and VAN A~PERENK. (1962) Partial purification and properties of fiyhead cholinesterase. J. Ins. Physiol. 8, l-14. ELLMAN G. L., COURTNEYK. D., ANDRES V., and FEATHERSTO~JE R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity, Bioc&. Phannacol. 7, 88-95. GORNALL A. G., BARDAWXLLC. J., and DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction, J, biol. Chem. 177, 751 -766. HEATH D. F. (1961) O~gunophosphorus Poisons, pp. 103-115. Pergamon Press, New York. JOHNSONM. J. (1949) A rapid micromethod for estimation of non-volatile organic matter. J. biol. Chem. 181, 707-711. LADLER H. C. (1959) A simplified procedure for the partial purification of acetylcholinesterase from electric tissue. J. bioi. Chem. 234, 799-801. LADLER H. C. (1961) Structural aspects of a~~icho~ester~. Fed. P~oc. 20, A-381f. Lozn K. A. (1961) The partial purification and properties of a cholinesterase from Bkzttdla germanica L. Bi0them.J. 78,4834%. METCALFR. L. and MARCHR. B. (1949) Studies on the mode of action of parathion and its derivatives and their toxicity to insects. J. econ. Ent. 42, 721-728. METCALF R. L. and MARCH R. B. (1950) Properties of acetylcholinester from the bee, the fly, and the mouse; and their relation to insecticide action. J. econ. Ent. 43.670-677. METCALF R. L., MARCH R. B., and MAXON M. G. (1955) Substrate properties of insect cholinesterasea. Amr. ent. Sac. Amer. 48, 222-228. MICHEL H. 0. (1949) An electrometric method for the determination of red blood cell and plasma chohnesterase activity. J. Lab. c&n. Med. 34,1564-1568. MOOREFIELDH. H. (1957) Improved method of harvesting house fly heads for use in cholinesterase studies. Contr. Boyce Thompson Inst. 18, 463. MORTON R. K. (1950) Separation and purification of enzymes associated with insoluble particles. Nature, Lomb. 166, 1092-1095. ROTHENBERGM. A. and NACHMANSOHN D. (1947) Studies on cholinesterase.-III. Purification of the enzyme from electric tissue by fractional ammonium sulfate precipitation. J. biol. Chem. 168, 223-231. SMALLMAN B. N. and WOLFE L. S. (1956) Soluble and particulate cholinesterase in insects. y. cell. camp. Fhysiol~ 48, 197-213. WILSON I. B. (1960) Acetylchohnesterase. In The Enzymes (Ed. by BOYER P. D., LAY H., and MYRBXCKK.), Vol. 4, 501-520. Academic Press, New York. WOLFE L. S. and SMALLMAN B. N. (1956) The properties of cholinesterases from insects. J. cell. camp. Physiol. 48, 215-235.