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Effect of salt on the hydrolysis of acetylcholine by cholinesterases
Effect of Salt on the Hydrolysis of Acetylcholine by Cholinesterases David K. Myers From the Pharmaco-Therapeutic Laboratory of the University of Amsterdam, Amsterdam, Holland Received
December
12, 1951
It has been shown by several investigators that the cholinesterase activity of mammalian brain and erythrocytes exhibits a definite maximum at low concentrations of acetylcholine and is strongly inhibited by an “excess” of acetylcholine (1,2,3). This fact has been frequently confirmed and is generally accepted although there is some disagreement as to the exact value of the optimum concentration of acetylcholine (Ach). It is known now that the tissue preparations used by these invest,igators contain mainly true cholinesterase (true ChE) with litt#le or no pseudocholinesterase present (2,4) and that this inhibition by excess Ach is characteristic of mammalian true ChE. On the other hand, the activity of the pseudocholinesterase (pseudo-ChE) reaches its maximum only at very high concentrations of Ach and does not show any evidence of inhibition by excess Ach (2,4). The experimental results obtained with an enzyme: substrate system such as the pseudo-ChE: Ach system are in good agreement with the theory that enzyme activity is due to activation of the substrate by the formation of an active complex between the substrate and the enzyme active center. Murray (5) and Haldane (6) had proposed that the phenomenon of excess substrate inhibition might be explained by assuming that an inactive combination is formed between the enzyme active center and two molecules of substrate at high substrate concentrations. On the basis of this theory, Zeller and Bissegger (3) postulated that the active center of the true ChE molecule might be essentially dipositional in nature, one position being an “ester grouping” which combines with the ester linkage of the substrate, and the second position being an adjacent anionic grouping which combines with the cationic ammonium group of Ach. At low concentrations of Ach, 469
470
DAVID
K. MYERS
these two positions could combine with the ester and cationic groups of the same molecule of Ach to give the usual active complex between one molecule of substrate and one molecule of enzyme. In the presence of excess Ach, the ester grouping might combine with the ester linkage of one Ach molecule and the anionic grouping with the cationic ammonium group of a second Ach molecule to give an inactive complex The Haldane theory would predict a symmetrical bell-shaped curve when the activity of an enzyme subject to excess substrate inhibitSion is plotted graphically against the logarithm of the substrate concent#ration. The theoretical equations concerned were applied by Augustinsson (7) to the excess substrate inhibition of true ChE by Ach, and the experimental results were found to correspond fairly well with the symmetrical bell-shaped curve expected. This might be taken as substantial evidence in favor of the hypothesis of Zeller and Bissegger (3), and the same theory of a dipositional enzyme center for true ChE also provides a good explanation of the effects of salts (8,9) and pH (10,ll) upon the affinity of various inhibitors for the true ChE. The effect of salts upon the hydrolysis of Ach by t,rue ChE was investigated by Mendel and Rudney (12). Their results and those of Alles and Hawes (1) show that the addition of salts to the medium decreases the affinity of the true ChE for Ach with a consequent shift in the optimum concentration of Ach to higher values, and also gives an absolute increase in the rate of Ach hydrolysis at the new optimum levels. Under certain conditions, a competitive inhibitor could cause effects of this nature, as will be described later. The presence of salts also decreases the affinity of the ChE’s for eserine, prostigmine, and analogs with a cationic ammonium group (8,9). It appeared in this case that the cation of the salt was itself acting as a reversible inhibitor which competed for t#heanionic position in the ChE act,ive center. Thus it was of interest to reinvestigate the effect of salts upon the hydrolysis of Ach by ChE with the purpose of determining how these results could be interpreted in terms of the Murray-Haldane theory and to ascert,ain whether the salts were acting simply as competitive inhibitors of the true ChE. METHODS Cholinesterase activity was determined manometrically by the Warburg method as in previous investigations (2,4,8,9,12). The fluid medium contained 0.025 ik! sodium bicarbonate in all cases and was saturated with 5y0 CO, in Nz to give a pH of 7.4 at 37.5”C. Dialyzed hemolyzed human erythrocytes were used as the source of true cholinesterase and dialyzed human serum as the source of pseudocholinesterase.
HYDROLYSIS
OF
ACETYLCHOLINE
471
True ChE activity at very low concentrations of acetylcholine was determined by the same technique as used by Mendel and Rudney (12). This technique has been criticized several times by Augustinsson and Nachmansohn (7,13,14,15) but has given reliable results in the present investigation. These latter authors were unable to confirm Mendel and Rudney’s results but it seems possible that this might be ascribed to the use of different methods. The following points are of special importance in t.his regard: (a) The enzyme preparation is suspended in a large volume of buffer solution with or without the other salts; after a suitable period of incubation in the Warburg vessel, a small volume of substratp solution is added from the side arm (2,12). In Augustinsson’s experiments (7), it would appear that a small volume of enzyme and salt solution were placed in the side arm of the Warburg vessel and then mixed with a relatively large volume of substrate solution when the enzyme activity was to be measured. This, of course, entails a considerable dilution of the salt and is not an advisable technique with any reversal inhibitor where the dilution will affect the degree of inhibition obtained. The inhibitory effects of salts especially are very quickly reversed after dilution of the salt concentration and addition of substrate. (5) The total volume of fluid used by Mendel and Rudney (12) was 6.0 ml.; that used by Augustinsson (7) was 2.0 ml. At low concentrations of acetylcholine, the reaction time is limited by the total amount of substrate available; for example, 6 ml. of a 4 mg.-y0 (2.5 X lo+ M) Ach solution will give a total of 34 ~1. CO1 at complete hydrolysis of the Ach. This is sufficient to permit a fairly accurate measurement of the constant initial rate of hydrolysis when the enzyme activity is about 7 J. CO&nin. and the readings are made at l-mm. intervals. When a smaller volume of solution is used, similar determinations can only be made at higher concentrations of Ach (7). If the readings are not made at l-mm. intervals but at IO-min. intervals as in the investigations of Zeller and Bissegger (3), it is quite possible that the substrate may have been almost completely hydrolyzed long before the first reading is made. Under such conditions the figures obtained may not bear any relationto the initial constant velocity rate which is the most suitable measure of enzyme activity. (c) The acetylcholine in the control and experimental vessels is tipped into the main compartments of the respective vessels simultaneously, and readings are taken immediately after re-establishment of thermal equilibrium (12) ; the process of tipping should not take more than 4-5 sec. The enzyme activity is determined by comparison of the experimental vessel with a control vessel which was tipped simultaneously to compensate for any lack of thermal equilibrium, but this control does not alter appreciably when the readings are begun 1 min. later. The results obtained with low concentrations of Bch are illustrated by a typical protocol given in Table I; it is apparent that thermal equilibrium has been quickly re-established after tipping the vessels. The criticisms of Augustinsson and Nachmansohn (7,13,14,15) are not applicable to this technique. (d) The experiments of Mendel and Rudney (12) have been criticized on the basis that they were not carried out under optimal conditions in regard to the salt concentration (14). A small amount of buffer is always necessary to maintain a constant pH in these experiments but otherwise the cholinesterases of mammalian tissues do not seem to require the presence of large concentrations of salts for their activity. Thus this criticism is rather difficult to understand since the essence of this experiment is
472
DAVID
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TABLE Experimental
Control
Protocol Illustrating the Hydrolysis of Low Concentrations by the True ChE of Human Erythrocytes, in the Presence and in the Absence of 0.5 N NaCl
veseel:
Experimental with buffer
x 10-p 111 Sch in side srm
Time min.
I of .4ch
vessels: 0.1 ml. erythrocyte hemolyzste in main compartment solution; 0.06 ml. of 2.5 X 10-s M (0.4%) Ach solution in side arm (final concn. in volume of 5 ml. = 3 X 10-k M Ach)
0.025
M bicarbonate
Change in manometer reading
Bicarbonate
+ 0.5 M NsCI
2”. activity
?!Y activity
Before mixine: enzvme with substrate 15-18 I 18-21 I
0.5 0.0
After l- 2 2- 3 3- 4 4-5 5- 6 6- 7 7- 8 8- 9 9-10 lo-11
mixina
I 1
0.5 0.0
I I
0.0-I” 0.0 1
enzvme with substrate I
0.5 0.0 0.0 -0.5 0.0 0.5 0.0 0.0 -0.5 0.0
4.5 5.0 4.5 1 4.0 3.0 2.5 1.5 0.5 0.5 0.0
5.0 5.0 4.5 3.5 3.0 3.0 1.5 0.5 0.0 0.0
I 1.0 1 -0.5
0
I 0.5 1 -0.5
I 1
in all vessels simultaneouslv ---I
14.0 x 1.41a = 19.8 pl./ 3 min. = 132 pl./ 20 min.
3.0 2.5 2.0 2.0 3.0 3.0 2.0 2.5 1.5 1.5
2.5 2.5 2.0 2.5 3.0 2.5 2.0 2.5 2.0 1.5
0
at 0 min. 19.5 x 1.49” = 29.1 /.&I./ 8 min. = 72.8/J./ 20 min.
r-
The same as above but with 0.2 ml. of 2.5 X lo* M Ach to give a final concentration of 1 X lo+ llf Ach Before mixing enzyme with substrate 15-18 18-21 After l- 2 2- 3 3- 4 4- 5 5- 6 6-7 7- 8 8-9 9-10 lo-11
0.0 mixing
1
:.x
enzyme with
0.0 0.5 0.0 0.5 0.5 -0.5 0.0 -0.5 0.0 0.5
0 Vessel constant
4.5 5.5 4.5 5.0 5.0 4.5 4.5 4.0 4.0 4.0
I
x::
I
substrate 4.5 5.0 I 4.5 4.5 4.5 5.0 4.5 4.5 4.0 3.5
for converting
0
I -x:i
I -8::
in all vessels simultaneously 37.0 x 1.510 = 55.9pl./ 8min. = 139.6,&/ 20 min.
manometer
4.5 4.5 4.5 5.0 4.5 4.5 4.5 4.0 4.0 4.0
4.5‘ 4.0 4.5 4.5 4.0 5.0 4.5 4.5, 4.0 3.5
reading to pl. CO*.
1
0
at 0 min. 35.5 x 1.52” = 54.Opl./ 8 min. = 135rl./ 20 min.
HYDROLYSIS OF ACETYLCHOLINE
473
to compare the true ChE activity towards Ach at low salt concentrations and at high salt concentrations. If the medium already contains relatively large amounts of salts (7,13), the addition of further small amounts of salt will not have much effect. THEORETICAL
CONSIDERATIONS
According to the classical theory of enzyme action as proposed by Michaelis and Menten (16), the activity of an enzyme is directly proportional to the concentration of a complex between enzyme and substrate. The reversible reaction between free enzyme (Kf) and substrate (~5’)is represented as
The mass law equation for a reversible equilibrium of this kind would be Ef x S = K,, where K. is the so-called “dissociation” or Michaelis constant which is ES inversely proportional to the affinity of the substrate for the enzyme. If we put the fractional activity a = ES/E and (1 - a) = Ef/E (16), then S = K,&ori
= l+%.
This equation is applicable to the usual enzyme reactions which are not subject to excess substrate inhibition, as for example the hydrolysis of Ach by pseudo-ChE. The usual interpretation placed upon the Murray-Haldane theory of excess substrate inhibition (5,6) is to picture the reaction between enzyme and substrate as follows: Then -EJXS ES
=
K
ES x S a, and Es2 = K.,.
In this case (1 - a) = (El+ E&)/K,
and
1 -=1+x a
K.1 +g a*
(2)
or, as it is more frequently written (5,7), K.,S a = S4 + K,,S + K,,K.,. It is often advantageous in this case to use the older terminology where the fractional activity a = v/Vm,, v being the velocity of the enzyme action at any given substrate concentration and V,, the theoretical maximal velocity. We could also picture the above reactions occurring as follows: Ef+Si=
ES, and El + 25 s ES?.
This would give the theoretical equation (3)
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However, this does not affect the shape of the symmetrical bell curve in any way. The difference between Eqs. (2) and (3) lies in the magnitude of K,,, that is to say, the same bell-shaped curve will be obtained when K,, in Eq. (3) is K,, times as large as the constant K,, in Eq. 2.
FIG. 1. Theoretical curves showing the relationship between the fractional activity “a” and the negative logarithm of the substrate concentration as calculated from Eq. (2). K,, = lo+ M in all cases; K., = 1 X 10m4 11f, 3 X lo-” M, 1 X 1O-3 kf, 3 X 10m3 ilr and 1 X times 1O-2 M for the curves in order from left to right. Inset: Experimental curves obtained by Mendel and Rudney (12) for the hydrolysis of Ach by the true ChE of mouse brain in the presence of increasing concentrations of KCI. Ordinate is the enzyme activity as d. CO2/12 min., abscissa is the negative logarithm of the molar concentration of Ach. If we give K., any arbitrary value and calculate out the relationship between a and log S from Eq. 2, a series of curves is obtained, as shown in Fig. 1. In general, whenever a competitive inhibitor of concentration Z is equilibrated with an enzymesubstrate
system, the Michaelis
constant
K, will appear to be increased by
HYDROLYSIS
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ACETYLCHOLINE
times. In the case of an enzyme subject to excess substrate inhibition, it is conceivable that a selective inhibitor might have more effect upon the apparent value of K,, than upon that of K,,. Under these conditions not only would the optimum substrate concentration be shifted to a higher level but the absolute rate of hydrolysis at the new opt,imum level would be higher than that at the original optimum concentration of
0’
5
4
3
2
I
FIG. 2. Theoretical curves showing the relationship between the fractional activity “a” and the negative logarithm of the substrate concentration as calculated from Eq. (4) assuming y = 0.1. K,, = 1O-4 111in all cases; Kd? = 1 X lo-* IIf, 4 X 10-d di, 1 X 10-J &I, 4 X 1O-3 iW, 1 X lOma LIT, and 4 X 10e2 111for the curves in order from left to right. substrate. The superficial similarity between such curves and those given by Mendel and Rudney (12) is apparent from Fig. 1. The results of Alles and Hawes (l), Mendel and Rudney (12), and Augustinsson (7) show that the a vs. log S curves are somewhat asymmetrical in most cases with the ChE’s. One explanation of this asymmetry might be that the complex ES, is not completely inactive. This does not seem unreasonable in view of the fact that a substrate such as methyl acetate is hydrolyzed slowly by the true ChE (9) although it
476
DAVID K. MYERS
would combine only with the ester grouping and not with the anionic grouping nor with other substituent groupings which would combine with the choline moiety of the Ach molecule. Similarly we might suppose that an Ach molecule which is attached to the true ChE only by the acetate moiety might also be subject to slow hydrolysis. We could postulate that the ratio of activity of ES2 to activity of ES is equal to a certain fraction
y. Then the total fractional
activity
a should be equal to 7
+ y T.
E’&=E-ES-E,=E-ES(l+%) and substituting
the value of ES/E from Eq. 2, then
ES2 -= E
Thus, for the circumstances
a=T+y+
sz Se + K.,S + KJL,’
outlined
above,
K,,S + YS= P f K,,S + K.,K.,’
(4)
If we assume that y = 0.1, for example, and construct a series of curves analogous to those shown in Fig. 1, we obtain the asymmetric bell-shaped curves of Fig. 2. As the substrate concentration is increased to very high values, a point should be reached at which the enzyme activity becomes minimal; further increases in the substrate concentration should not affect the activity significantly. It did not always prove possible to determine whether this criterion was fulfilled in the case of excess substrate inhibition of the ChE’s but in many cases it was essential to use Eq. (4) to obtain a theoretical curve which would fit the experimental results.
EXPERIMENTAL
RESULTS
The general type of results obtained by Mendel and Rudney (12) with the true ChE of mouse brain was readily confirmed in the present investigation with the true ChE of human erythrocytes. This is illustrated by the protocol given in Table I. It can be seen that there is very little change in the true ChE activity when the concentration of Ach is increased from 3 X lo4 M to 1O-3 M in a 0.025 M sodium bicarbonate medium, which would indicate that the optimal concentration of Ach lies in this range. In the presence of 0.5 M sodium chloride, the true ChE activity at both concentrations of Ach is considerably lower than in the presence of 0.025 M bicarbonate alone; further, the true ChE activity increases markedly as the concentration of Ach is increased from 3 X 10” M to 10m3M indicating that the optimum must lie at a considerably higher range of concentrations under these conditions. The true ChE activity of the erythrocytes was similarly determined with a series of Ach concentrations ranging from 3 X lo4 M (5 mg.-%)
HYDROLYSIS
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ACETYLCHOLINE
477
to 0.2 M (3.3%) with an increase in concentration of approximately 1.5 times at each step of the series. The results were plotted graphically against the logarithm of the substrate concentration and the best theoretical curve was fitted to these points by a series of trials with decreasing error. It was found that the results obtained with a 0.025 -U bicarbonate medium could not be fitted by any of the symmetrical bell-shaped curves derived from Eq. (2). Different forms of the asymmetrical curves given by Eq. (4) were then tried; the theoretical curves for which y = 0.1 proved to give a relatively good fit to the experimental results. This value of y was also assumed for the other experimental curves where the value of y could not be accurately determined (e.g., in the presence of 0.2 and 0.5 M NaCI). The experimental results correspond fairly well to these curves in all cases but would also correspond to theoretical curves in which y < 0.1. However, the values of V,., and K,, would hardly be affected by the assumption of a smaller value for y in the presence of higher concentrations of sodium chloride, and the error introduced in the estimations of K,, would be relatively small. The general trend of the results will not be affected by this assumption. The curves obtained are presented in Fig. 3; it can be seen that these curves resemble closely the experimental curves given by Mendel and Rudney (12). Most of the values of K,, and K,, can be determined with some accuracy from the experimental results except in the case where a 0.025 211bicarbonate medium is used. The lowest concentration of Ach at which the activity can be tested is limited to about 3 X lo4 M by this technique, and the true ChE activity in a 0.025 A1 bicarbonate medium shows very little change with changes in the Ach concentration between 3 X 10m4J1 and 2 X 1O-3 M. However, the average of several series of experiments indicated that the activity at 6-8 X 1O-4 ikl was slightly higher than the activities at either lower or higher concentrations of Ach. Thus a theoretical curve was drawn with an optimum at approximately 7 X lop4 AT but this value must be regarded as somewhat indefinite. The shift in the optimum concentration after addition of as little as 0.02 M NaCl or 0.005 M CaCl* was quite definite nevertheless. The addition of increasing concentrations of sodium chloride to the medium caused a progressive increase in the values of both K,, and K,, (Fig. 3). The value of K,, was increased tenfold by the addition of 0.5 M sodium chloride to a 0.025 M bicarbonate medium, whereas the
478
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MYERS
I GO
I a.0
.L -9 0
80
40 20
-logs FIG. 3. Experimental results obtained for the hydrolysis of Ach by the true ChE of human erythrocytes in the presence of various concentrations of NaCl increasing in order from left to right. The ordinate gives the enzyme activity as 4. COJO.1 ml. erythrocyte hemolyzate/20 min., the abscissa gives the negative logarithm of the molar concentration of Ach. The theoretical curves were calculated from Eq. (4) assuming y = 0.1; the best theoretical curves were obtained using the following values:
0.55 1.10 200 0.78 0.021 1.10 1.21 110 1.15 0.05 1.9 1.35 70 1.60 0.10’ 2.6 1.55 60 2.05 0.20 3.7 1.8 50 2.70 0.50 5.5 2.2 40 3.65 1 To avoid unnecessary confusion on the graph, these two curves in Fig. 3.
140 157 146 169 156 190 168 208 175 221 171 221 were not included
HYDROLYSIS
OF
ACETYLCHOLINE
479
value of K,, was increased only by twofold so that the ratio of K,, to K,, becomes progressively smaller. The optimum concentration of Ach is also shifted to higher values as shown by Mendel and Rudney (12). The absolute rate of hydrolysis at the new optimum level was increased
2oo I-
Fro. 4. Experimental results obtained for the hydrolysis of Ach by the true ChE of human erythrocytes in the presence of 0.025 M sodium bicarbonate alone (left) and with 0.005 M calcium chloride added (right). The theoretical curve for activity in the 0.025 M bicarbonate medium is the same as that given in Fig. 3; the curve for the activity in the presence of CaClz was calculated on the same basis using the following values: K., = 2.75 X 1O-4 M Ach, K,, = 1.10 X lO-e M Ach, K.,/K., = 40, f&t = 1.9 X 10m3III Ach, V,,t = 186 ~1. COJ20 mm., Vu, = 241 ~1. CO*/20 mm.
227c by the addition of 0.5 M NaCl, but the theoretical maximum was increased even more (41%). This is because the progressive decrease in the ratio K,,/K,, tends to mask this activation to some extent. The activation of the true ChE activity toward Ach is even more
480
DAVID
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apparent with a bivalent salt such as calcium chloride. In this instance a special technique was necessary since a slow reaction liberating free CO, occurs when a bicarbonate medium containing CaClz is incubated at 37”. In the presence of erythrocyte hemolyzate the calcium ion is apparently bound by the proteins and the reaction does not occur (17). The controls for this experiment contained erythrocyte hemolyzate
200
-
180
-
IGO I40
-
120
-
100
-
8o 60
-
40
-
> -$ .-
2
FIG. 5. Experimental results obtained for the hydrolysis of Ach by the pseudo-ChE of human serum in the presence of 0.025 M sodium bicarbonate alone (left) and with 0.5 M sodium chloride added (right). The ordinate gives the enzyme activity as ~1. COP/O.1 ml. serum/20 min.; the abscissa gives the negative logarithm of the molar concentration of Ach. The theoretical curves were calculated from Eq. (1) using the following values: Medium
K.
hW.X
0.025 M NaHCO, + 0.5 M NaCl
1.38 x 10-s M Ach 2.68 x 10-S M Ach
206 ~1. CO*/20 min.
175 j4l. CO&Kl min.
HYDROLYSIS
OF
ACETYLCHOLINE
481
with 10m6M diisopropyl fluorophosphonate added to inhibit completely the true ChE activity toward Ach. The results obtained by this method are shown in Fig. 4. In contrast to the effects of NaCl, 0.005 211CaClz appeared to have very little effect upon the value of K,, even though the value of K,, was increased markedly. The optimum concentration of Ach is also shifted to higher levels, but the most striking effect of the small concentration of CaClz is the strong activation of the maximal activity of true ChE toward Ach. It is usually assumed that salts have little effect upon the hydrolysis of Ach by pseudo-ChE but it was possible to demonstrate a slight shift in the affinity constant in the present investigation when high concentrations of sodium chloride were employed. This agrees with previous experiments in which it was shown that the presence of sodium and potassium chloride does inhibit the activity of pseudo-ChE towards suboptimal concentrations of Ach (9,18). Determinations of pseudoChE activity with a series of different concentrations of Ach showed that the affinity of pseudo-ChE for Ach was significantly decreased (1.9 times) by the addition of 0.5 M NaCl to the medium (Fig. 5), although the decrease in affinity (or increase in K,) is not nearly as striking as in the case of the true ChE. The experimental results could be fitted quite well by a simple equation of form 1. It appeared that the maximal activity wasPlightly lower in the presence of salt than in its absence, an effect which is not dependent on the increase in K,. This would indicate that the salt acts not only as a simple reversible competitive inhibitor (K, increased, V,, unchanged) but also as a noncompetitive inhibitor (K, unchanged, V’,,, decreased). DISCUSSION
It has been shown that the maximal activity of the true ChE toward Ach is considerably potentiated by the presence of salts, and especially by a bivalent salt such as calcium chloride. However there has not been any conclusive evidence to indicate that the presence of salts is essential for the activity of either the true ChE or the pseudoChE’s of mammalian tissue. There is very little loss in activity when the true ChE of hemolyzed erythrocytes or the pseudo-ChE of serum is subjected to ordinary dialysis against distilled water for 24 hr. as in the present and previous (8,9) investigations. It is essential that the pH should be in the neighborhood of 7, and it is preferable that the dialysis
482
DAVID
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MYERS
should be carried out at. a low temperature (3°C.) t,o prevent inactivation. This might appear to contrast with the results of Nachmansohn et al. (19,ZO) who found that the true ChE of the electric organ of Torpedo vulgaris was inactivated to a large extent, by the process of dialysis. However, these investigators concluded at the time that much of the inactivation was caused by the increased sensitivity of the dialyzed enzyme to cont,aminating traces of heavy metals and of copper in particular. They showed that the true ChE activity was dependent upon t’he presence of free sulfhydryl groups which were readily inactivated by the metallic impurities. It was concluded in a later investigation that the addition of bivalent cations (e.g., Gaff, Mg++, &In++) gave a reactivation of the dialyzed enzyme which was independent of the reactivation of sulfhydryl groups (21). It seems possible that this might be due in part to a shift in the optimum concentration of Ach to higher values in the presence of salt. When this work (21) was done, it was not known that these were two types of ChE and that the true ChE exhibits the phenomenon of excess substrate inhibition by Ach. The concentration of Ach employed was fairly high (0.016 M) and the enzyme would be strongly inhibited by the excess substrate since the activity-substrate concentration relationship with this enzyme preparation in the presence of 0.025 M bicarbonate is very similar to t,hat shown by the true ChE’s from mammalian tissues.2 Addition of salts to the medium would lead to a shift in the optimum concentration of Ach to much higher levels, and this in itself would appear to result in a large activation of the true ChE activity at a high concentration of Ach. This may not be a complete explanation of the above-mentioned results (Zl), but a full interpretation of the results with this enzyme preparation is impossible without a full picture of the enzyme activity at a series of Ach concentrations (22). It does seem possible by analogy with other enzymes that the presence of a very small amount of a bivalent cation may be essential for the activity of the ChE’s (23), but the experimental evidence has not been convincing. Bivalent salts do cause an appreciable activation of the pseudo-ChE (18) and true ChE activity, but prolonged dialysis has very little effect upon the activity of a crude or a highly purified preparation of pseudo-ChE (23, 24, 25). Moreover the results described ppOl~~~~~, B., AND RUDNEY, H., unpublished.
HYDROLYSIS
OF
ACETYLCHOLINE
483
above show that a simple salt such as sodium chloride also possesses the property of activating the true ChE albeit to a much smaller extent than calcium chloride. It has been suggested that the bivalent cation may be very strongly held by the ChE itself under the conditions of the dialysis (23) since the ChE activity is partially inhibited by substances which form insoluble salts with Ca++ (e.g., fluoride, oxalate, citrate, pyrophosphate) when present in high concentrat’ions (23). This latter fact would seem to substantiate the hypothesis that Ca++ or a similar ion does form an essential part of the ChE molecule if it is assumed that the action of these compounds upon ChE is due solely to the removal of bivalent cations from the solution. The reasons for the failure of some other investigators to confirm the shift in optimal Ach concentration for true ChE caused by the addition of salts have been discussed previously. The results obtained in the present investigation are in good agreement with the results of Alles and Hawes (1) and Mendel and Rudney (12). L411esand Hawes (1) used a t,itrimetric technique and were able to determine the true ChE activity accurately at much lower concentrations of Ach than is possible with the manometric technique used above. Theoretical equations of the same type as used above were fitted to their experimental data to give the curves illustrated in Fig. 6. It would appear from these curves that K,, = 2 X lop6 211Ach and K,, = 8 X 1O-3 ill -4ch for the true ChE of erythrocytes in the presence of 0.0059 111 NaCl. These values fit very well into the series of values obtained in the present investigation and offer substantial confirmation of the above results. The data of Mendel and Rudney (12) were concerned especially with the optimum concentration of Ach in the presence of various concentrations of salt and do not cover a sufficiently wide range of Ach concentrations to allow an accurate determination of K,, and K,, from the experimental data. However, a comparison of the optimal concentration of Ach and the activity at each optimum level (V,,,) shows a close agreement between their results and those obtained above (Table II). The one point of significant discrepancy is with regard to the optimal concentration of Ach in the presence of 0.025 JU sodium bicarbonate alone, the values obtained being 2.5 X lo-’ -V ,4ch and 7.8 X 1O-4 iW ilch in the two investigations, respectively. However, the true ChE’s employed were from very different sources (mouse brain and human erythrocytes). Moreover, the activity-substrate concentration curves
484
DAVID
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MYERS
are relatively flat in this concentration range so that the precise value of the optimum concentration cannot be accurately determined. The results obtained in the present investigation indicate that sodium chloride has two distinct actions upon the true ChE activity toward Ach. In the first place, as suggested previously, it acts as a reversible competitive inhibitor and causes an apparent increase in the
I.0
0.8 > 4-J ..-> d 0.G 0.4
0.2
results obtained by Alles and Hawes (1) and the corresponding FIG. 6. Experimenta theoretical curves used in the present investigation to fit these results. The ordinate is true ChE activity towards Ach expressed as milliliters of 0.02 N NaOH/20 mm.; the abscissa is the negative logarithm of the molar concentration of Ach (calculated from the experimental curves of Alles and Hawes where the concentration was expressed as miUimoles of Ach per 25 ml. of solution). The theoretical curves were cdculated from Eq. (4) assuming y = 0.1 and using the following vaiues:
0.0059 0.145
0.20 1.0
0.80
0.5
1.0
LO
0.81 1.32
values of K+ and of K,,, especially of the former. This leads to an increase in the optimum concentration of Ach. The second effect is an activation of the true ChE. This is probably similar to the effects of salt as previously described for the hydrolysis of Ach by the true ChE of Planaria (26) and for the hydrolysis of non-choline esters by the true ChE of human erythrocytes (9). It has been suggested previously that
HYDROLYSIS
OF
485
ACETYLCHOLINE
the activation by salts may be due to an interaction with the ester grouping of the true ChE active center (9). It is not clear how this could be correlated with the supposition that Ca++ (or a similar cation) forms an essential part of the active center, or with the theory (11) that the ester grouping of the active center contains a latent basic group and a latent acid group in its active condition. Like most other proteins, the true ChE molecule as a whole (or, at least, the particulate matter with which the true ChE is associated) possesses a negative charge at physiological pH (2T), and it is probable that this charge would be affected not only by alterations in pH but also by alterations in the concentration of salts in the medium. It is very difficult to see how this latter fact alone could explain the selective effects of salts upon ChE activity and inhibition [cf. Refs. (2,8,9,12,21,24,28)-J; it seems necessary to assume that the salts also have a specific effect upon the enzyme active center. TABLE II Comparisonof the Data of MeruZel and Rudney (1.2) and Those of the Present Znvestigatim Illustrating the Effect of Salts upon the Hydrolysis of Ach by True ChE Data of Made1
and R~;~neney(12)
Medium
0.025 M +0.04 $0.08 +0.16
NaHCOs M KC1 M KC1 M KC1
V0pt
0.25 1.5 2.0 3.0
100 118 122 125
Data from present investigation &t Medium
0.025 M +0.05 +O.lO +0.20
NaHCOs M NaCl M NaCl M NaCl
0.78 1.60 2.05. 2.70
100 111 120 125
The evidence in favor of a dipositional active center for the true ChE has been critically reviewed recently by Whittaker (29). Three groups of investigators working independently from three different approaches have produced evidence for the existence of an anionic position in the enzyme active center (8,10,30). It would be expected that the anionic position has the effect of increasing the affinity of the true ChE for Ach and analogous esters and of facilitating the hydrolysis of the ester linkage by the esterasic site (29), or “ester grouping.” In so far as the salt acts as a reversible competitive inhibitor of true ChE, we could postulate as in the previous investigations (8,9) that the cation competes with Ach for the anionic position in the ChE active center. The addition of sodium chloride to the medium decreased the affinity
486
DAVID K. MYERS
of Ach both for the true ChE and for the pseudo-ChE. It would follow that the active center of the pseudo-ChE as well as that of the true ChE is probably dipositional in nature. However the combination of Ach with the pseudo-ChE does seem to be much less dependent upon the cationic ammonium group of Ach than is the case with the true ChE. The experimental results obtained previously with t#he true ChE (8) show that the addition of 0.475 M NaCl to a bicarbonate medium decreased the affinity for eserine by sixfold (-log 160 decreased by 0.78). The above results indicate that 0.5 M NaCl decreases the affinity of the true ChE for Ach by even more, i.e., tenfold (K,, increased ten times). For the pseudo-ChE, on the other hand, the addition of 0.5 M NaCl resulted in a change of 5.2 times (antilog 0.72) with prostigmine (9), 3.8 times (antilog 0.58) with eserine (9), and 1.9 times with Ach. This could be interpreted as meaning that the affinity of the pseudo-ChE for Ach is much less dependent upon the anionic position of the enzyme active center than is the case with eserine or prostigmine. The reason for the difference between Ach and prostigmine is not apparent, but the conclusion in itself does agree with the results of other workers who have shown that the binding of Ach by pseudo-ChE is mainly dependent upon the ester structure of the Ach molecule (30,31). ACKNOWLEDGMENTS The author is indebted to Dr. B. Mendel for his criticisms and suggestions, and to Misses M.. de Jonge and E. Simons for their technical assistance. SUMMARY 1. In confirmation of the results of Mendel and Rudney, it has been shown that the optimum acetylcholine concentration for true cholinesterase is shifted to progressively higher levels by the addition of increasing concentrations of salt to the medium. 2. The theoretical equations for Murray and Haldane have been applied to the resulting curves between enzyme activity and substrate concentration. It has been found that these equations will not fit the experimental results unless the assumption is made that the “inactive” complex ES, is subject to slow hydrolysis. The relative activity of the ES2 complex would appear to be about one-tenth of the activity of the ES complex between enzyme (E) and substrate (S). 3. The addition of simple salts (e.g., NaCI, KCl) decreases the affinity of true cholinesterase for acetylcholine. The salts have a further
HYDROLYSIS
OF ACETYLCHOLINE
487
effect in potentiating the hydrolytic activity of true cholinesterase toward acetylcholine; this activation is especially marked with a bivalent salt such as calcium chloride. The significance of these results has been discussed in relation to the theory of a dipositional enzyme active center. 4. The results support the supposition that the active center of the pseudocholinesterase is also dipositional in nature but the affinity of acetylcholine for pseudocholinesterase appears to be dependent mainly on the ester structure of the acetylcholine molecule. REFERENCES 1. ALLES, G. A., AND HAWES, R. C., J. Biol. Ch.em. 133, 375 (1940). 2. MENDEL, B., AND RUDNEY, H., Biochem. J. 37, 53 (!943). 3. ZELLER, E. A., AND BISSEGGER, -4., Helv. Ch.im. Acta 26, 1619 (1943). 4. MENDEL, B., MUNDELL, D. B., AND RUDNEY, H., Biochem. J. 37, 473 (1943). 5. MURRAY, D. R. P., Biochem. J. 24, 1890 (1930). 6. HALDANE, J. B. S., Enzymes. Longmans, London, 1930. 7. AUGUSTINSSON, K.-B., Beta Physiol. &and. Suppl. 15, 52 (1948). 8. MYERS, D. K., Arch. Biochem. 27, 341 (1950). 9. MYERS, D. K., Arch. Biochem. Biophys. 31, 29 (1951). 10. WILSON, I. B., AND BERGMANN, F., J. Biol. Chem. 185, 479 (1950). 11. WILSON, I. B., AND BERGMANN, F., J. Biol. Chem. 186, 683 (1950). 12. MENDEL, B., .&ND RODNEY, H., &%nce 102, 616 (1945). 13. NACAMANSOHN, D., AND ROTHENBERG, hI. A., J. Biol. Chem. 158, 653 (1945). 14. AUGUSTINSSON, K.-B., AND NACHMANSOHN, D., Science 110, 98 (1949). 15. AUGUSTINSSON, K.-B., The Enzymes, Vol. 1, p. 443. Academic Press, New York,
16. 17.
18. 19.
20. 21. 22.
23. 24.
25. 26. 27. 28. 29. 30. 31.
1950.
MICHAELIS, L., AND MENTEN, M. L., Biochem. 2. 49, 333 (1913). See KREBS, H. -4., 2. physiol. Chem. 217, 191 (1933); KREBS, H. A., AND EGGLESTON, L. V., Biochem. J. 34, 442 (19-U)). MENDEL, B., hIUNDELL, D. B., AND STRELITZ, F., Nature 144, 479 (1939). NACKMANSOHN, D., AND LEDERER, E., Compt. rend. sot. biol. 130, 321 (1939). NACHMANSOHN, D., AND LEDERER, E., Bull. sot. chim. biol. 21, 797 (1939). NACA~TANSOHN, D., Nature 145, 513 (1940). WYNNE, A. M., Ann. Rev. Biochem. 15, 35 (1946). MASSART, L., AND DUFAIT, R., Enzymologia 6, 282 (1939). MENDEL, B., MUNDELL, D. B., AND STRELITZ, F., Nature 145, 822 (1940). STRELITZ, F., Biochem. J. 38, 86 (1944). HAWKINS, R. D., AND hIENDEL, B., J. Cellular Comp. Physiol. 27, 69 (1946). AUGU~TINSSON, K.-B., Arkiv Kemi Mineral. Gwl. 18A, No. 24 (1944). MEER, C. VAN DER, Chem. It’eekblad, 48, 118 (1952). WHITTAKER, V. P., Physiol. Revs. 31, 312 (1951). ADA~G, D. H., AND WHITTAKER, V. P., Biochem. et Btiphys. Acta 4, 543 (1950). ELEY, D. D., AND STONE, G. S., Biochem. J. 49, XXX (1951).
December
12, 1951
It has been shown by several investigators that the cholinesterase activity of mammalian brain and erythrocytes exhibits a definite maximum at low concentrations of acetylcholine and is strongly inhibited by an “excess” of acetylcholine (1,2,3). This fact has been frequently confirmed and is generally accepted although there is some disagreement as to the exact value of the optimum concentration of acetylcholine (Ach). It is known now that the tissue preparations used by these invest,igators contain mainly true cholinesterase (true ChE) with litt#le or no pseudocholinesterase present (2,4) and that this inhibition by excess Ach is characteristic of mammalian true ChE. On the other hand, the activity of the pseudocholinesterase (pseudo-ChE) reaches its maximum only at very high concentrations of Ach and does not show any evidence of inhibition by excess Ach (2,4). The experimental results obtained with an enzyme: substrate system such as the pseudo-ChE: Ach system are in good agreement with the theory that enzyme activity is due to activation of the substrate by the formation of an active complex between the substrate and the enzyme active center. Murray (5) and Haldane (6) had proposed that the phenomenon of excess substrate inhibition might be explained by assuming that an inactive combination is formed between the enzyme active center and two molecules of substrate at high substrate concentrations. On the basis of this theory, Zeller and Bissegger (3) postulated that the active center of the true ChE molecule might be essentially dipositional in nature, one position being an “ester grouping” which combines with the ester linkage of the substrate, and the second position being an adjacent anionic grouping which combines with the cationic ammonium group of Ach. At low concentrations of Ach, 469
470
DAVID
K. MYERS
these two positions could combine with the ester and cationic groups of the same molecule of Ach to give the usual active complex between one molecule of substrate and one molecule of enzyme. In the presence of excess Ach, the ester grouping might combine with the ester linkage of one Ach molecule and the anionic grouping with the cationic ammonium group of a second Ach molecule to give an inactive complex The Haldane theory would predict a symmetrical bell-shaped curve when the activity of an enzyme subject to excess substrate inhibitSion is plotted graphically against the logarithm of the substrate concent#ration. The theoretical equations concerned were applied by Augustinsson (7) to the excess substrate inhibition of true ChE by Ach, and the experimental results were found to correspond fairly well with the symmetrical bell-shaped curve expected. This might be taken as substantial evidence in favor of the hypothesis of Zeller and Bissegger (3), and the same theory of a dipositional enzyme center for true ChE also provides a good explanation of the effects of salts (8,9) and pH (10,ll) upon the affinity of various inhibitors for the true ChE. The effect of salts upon the hydrolysis of Ach by t,rue ChE was investigated by Mendel and Rudney (12). Their results and those of Alles and Hawes (1) show that the addition of salts to the medium decreases the affinity of the true ChE for Ach with a consequent shift in the optimum concentration of Ach to higher values, and also gives an absolute increase in the rate of Ach hydrolysis at the new optimum levels. Under certain conditions, a competitive inhibitor could cause effects of this nature, as will be described later. The presence of salts also decreases the affinity of the ChE’s for eserine, prostigmine, and analogs with a cationic ammonium group (8,9). It appeared in this case that the cation of the salt was itself acting as a reversible inhibitor which competed for t#heanionic position in the ChE act,ive center. Thus it was of interest to reinvestigate the effect of salts upon the hydrolysis of Ach by ChE with the purpose of determining how these results could be interpreted in terms of the Murray-Haldane theory and to ascert,ain whether the salts were acting simply as competitive inhibitors of the true ChE. METHODS Cholinesterase activity was determined manometrically by the Warburg method as in previous investigations (2,4,8,9,12). The fluid medium contained 0.025 ik! sodium bicarbonate in all cases and was saturated with 5y0 CO, in Nz to give a pH of 7.4 at 37.5”C. Dialyzed hemolyzed human erythrocytes were used as the source of true cholinesterase and dialyzed human serum as the source of pseudocholinesterase.
HYDROLYSIS
OF
ACETYLCHOLINE
471
True ChE activity at very low concentrations of acetylcholine was determined by the same technique as used by Mendel and Rudney (12). This technique has been criticized several times by Augustinsson and Nachmansohn (7,13,14,15) but has given reliable results in the present investigation. These latter authors were unable to confirm Mendel and Rudney’s results but it seems possible that this might be ascribed to the use of different methods. The following points are of special importance in t.his regard: (a) The enzyme preparation is suspended in a large volume of buffer solution with or without the other salts; after a suitable period of incubation in the Warburg vessel, a small volume of substratp solution is added from the side arm (2,12). In Augustinsson’s experiments (7), it would appear that a small volume of enzyme and salt solution were placed in the side arm of the Warburg vessel and then mixed with a relatively large volume of substrate solution when the enzyme activity was to be measured. This, of course, entails a considerable dilution of the salt and is not an advisable technique with any reversal inhibitor where the dilution will affect the degree of inhibition obtained. The inhibitory effects of salts especially are very quickly reversed after dilution of the salt concentration and addition of substrate. (5) The total volume of fluid used by Mendel and Rudney (12) was 6.0 ml.; that used by Augustinsson (7) was 2.0 ml. At low concentrations of acetylcholine, the reaction time is limited by the total amount of substrate available; for example, 6 ml. of a 4 mg.-y0 (2.5 X lo+ M) Ach solution will give a total of 34 ~1. CO1 at complete hydrolysis of the Ach. This is sufficient to permit a fairly accurate measurement of the constant initial rate of hydrolysis when the enzyme activity is about 7 J. CO&nin. and the readings are made at l-mm. intervals. When a smaller volume of solution is used, similar determinations can only be made at higher concentrations of Ach (7). If the readings are not made at l-mm. intervals but at IO-min. intervals as in the investigations of Zeller and Bissegger (3), it is quite possible that the substrate may have been almost completely hydrolyzed long before the first reading is made. Under such conditions the figures obtained may not bear any relationto the initial constant velocity rate which is the most suitable measure of enzyme activity. (c) The acetylcholine in the control and experimental vessels is tipped into the main compartments of the respective vessels simultaneously, and readings are taken immediately after re-establishment of thermal equilibrium (12) ; the process of tipping should not take more than 4-5 sec. The enzyme activity is determined by comparison of the experimental vessel with a control vessel which was tipped simultaneously to compensate for any lack of thermal equilibrium, but this control does not alter appreciably when the readings are begun 1 min. later. The results obtained with low concentrations of Bch are illustrated by a typical protocol given in Table I; it is apparent that thermal equilibrium has been quickly re-established after tipping the vessels. The criticisms of Augustinsson and Nachmansohn (7,13,14,15) are not applicable to this technique. (d) The experiments of Mendel and Rudney (12) have been criticized on the basis that they were not carried out under optimal conditions in regard to the salt concentration (14). A small amount of buffer is always necessary to maintain a constant pH in these experiments but otherwise the cholinesterases of mammalian tissues do not seem to require the presence of large concentrations of salts for their activity. Thus this criticism is rather difficult to understand since the essence of this experiment is
472
DAVID
K.
MYERS
TABLE Experimental
Control
Protocol Illustrating the Hydrolysis of Low Concentrations by the True ChE of Human Erythrocytes, in the Presence and in the Absence of 0.5 N NaCl
veseel:
Experimental with buffer
x 10-p 111 Sch in side srm
Time min.
I of .4ch
vessels: 0.1 ml. erythrocyte hemolyzste in main compartment solution; 0.06 ml. of 2.5 X 10-s M (0.4%) Ach solution in side arm (final concn. in volume of 5 ml. = 3 X 10-k M Ach)
0.025
M bicarbonate
Change in manometer reading
Bicarbonate
+ 0.5 M NsCI
2”. activity
?!Y activity
Before mixine: enzvme with substrate 15-18 I 18-21 I
0.5 0.0
After l- 2 2- 3 3- 4 4-5 5- 6 6- 7 7- 8 8- 9 9-10 lo-11
mixina
I 1
0.5 0.0
I I
0.0-I” 0.0 1
enzvme with substrate I
0.5 0.0 0.0 -0.5 0.0 0.5 0.0 0.0 -0.5 0.0
4.5 5.0 4.5 1 4.0 3.0 2.5 1.5 0.5 0.5 0.0
5.0 5.0 4.5 3.5 3.0 3.0 1.5 0.5 0.0 0.0
I 1.0 1 -0.5
0
I 0.5 1 -0.5
I 1
in all vessels simultaneouslv ---I
14.0 x 1.41a = 19.8 pl./ 3 min. = 132 pl./ 20 min.
3.0 2.5 2.0 2.0 3.0 3.0 2.0 2.5 1.5 1.5
2.5 2.5 2.0 2.5 3.0 2.5 2.0 2.5 2.0 1.5
0
at 0 min. 19.5 x 1.49” = 29.1 /.&I./ 8 min. = 72.8/J./ 20 min.
r-
The same as above but with 0.2 ml. of 2.5 X lo* M Ach to give a final concentration of 1 X lo+ llf Ach Before mixing enzyme with substrate 15-18 18-21 After l- 2 2- 3 3- 4 4- 5 5- 6 6-7 7- 8 8-9 9-10 lo-11
0.0 mixing
1
:.x
enzyme with
0.0 0.5 0.0 0.5 0.5 -0.5 0.0 -0.5 0.0 0.5
0 Vessel constant
4.5 5.5 4.5 5.0 5.0 4.5 4.5 4.0 4.0 4.0
I
x::
I
substrate 4.5 5.0 I 4.5 4.5 4.5 5.0 4.5 4.5 4.0 3.5
for converting
0
I -x:i
I -8::
in all vessels simultaneously 37.0 x 1.510 = 55.9pl./ 8min. = 139.6,&/ 20 min.
manometer
4.5 4.5 4.5 5.0 4.5 4.5 4.5 4.0 4.0 4.0
4.5‘ 4.0 4.5 4.5 4.0 5.0 4.5 4.5, 4.0 3.5
reading to pl. CO*.
1
0
at 0 min. 35.5 x 1.52” = 54.Opl./ 8 min. = 135rl./ 20 min.
HYDROLYSIS OF ACETYLCHOLINE
473
to compare the true ChE activity towards Ach at low salt concentrations and at high salt concentrations. If the medium already contains relatively large amounts of salts (7,13), the addition of further small amounts of salt will not have much effect. THEORETICAL
CONSIDERATIONS
According to the classical theory of enzyme action as proposed by Michaelis and Menten (16), the activity of an enzyme is directly proportional to the concentration of a complex between enzyme and substrate. The reversible reaction between free enzyme (Kf) and substrate (~5’)is represented as
The mass law equation for a reversible equilibrium of this kind would be Ef x S = K,, where K. is the so-called “dissociation” or Michaelis constant which is ES inversely proportional to the affinity of the substrate for the enzyme. If we put the fractional activity a = ES/E and (1 - a) = Ef/E (16), then S = K,&ori
= l+%.
This equation is applicable to the usual enzyme reactions which are not subject to excess substrate inhibition, as for example the hydrolysis of Ach by pseudo-ChE. The usual interpretation placed upon the Murray-Haldane theory of excess substrate inhibition (5,6) is to picture the reaction between enzyme and substrate as follows: Then -EJXS ES
=
K
ES x S a, and Es2 = K.,.
In this case (1 - a) = (El+ E&)/K,
and
1 -=1+x a
K.1 +g a*
(2)
or, as it is more frequently written (5,7), K.,S a = S4 + K,,S + K,,K.,. It is often advantageous in this case to use the older terminology where the fractional activity a = v/Vm,, v being the velocity of the enzyme action at any given substrate concentration and V,, the theoretical maximal velocity. We could also picture the above reactions occurring as follows: Ef+Si=
ES, and El + 25 s ES?.
This would give the theoretical equation (3)
474
DAVID
K.
MYERS
However, this does not affect the shape of the symmetrical bell curve in any way. The difference between Eqs. (2) and (3) lies in the magnitude of K,,, that is to say, the same bell-shaped curve will be obtained when K,, in Eq. (3) is K,, times as large as the constant K,, in Eq. 2.
FIG. 1. Theoretical curves showing the relationship between the fractional activity “a” and the negative logarithm of the substrate concentration as calculated from Eq. (2). K,, = lo+ M in all cases; K., = 1 X 10m4 11f, 3 X lo-” M, 1 X 1O-3 kf, 3 X 10m3 ilr and 1 X times 1O-2 M for the curves in order from left to right. Inset: Experimental curves obtained by Mendel and Rudney (12) for the hydrolysis of Ach by the true ChE of mouse brain in the presence of increasing concentrations of KCI. Ordinate is the enzyme activity as d. CO2/12 min., abscissa is the negative logarithm of the molar concentration of Ach. If we give K., any arbitrary value and calculate out the relationship between a and log S from Eq. 2, a series of curves is obtained, as shown in Fig. 1. In general, whenever a competitive inhibitor of concentration Z is equilibrated with an enzymesubstrate
system, the Michaelis
constant
K, will appear to be increased by
HYDROLYSIS
OF
475
ACETYLCHOLINE
times. In the case of an enzyme subject to excess substrate inhibition, it is conceivable that a selective inhibitor might have more effect upon the apparent value of K,, than upon that of K,,. Under these conditions not only would the optimum substrate concentration be shifted to a higher level but the absolute rate of hydrolysis at the new opt,imum level would be higher than that at the original optimum concentration of
0’
5
4
3
2
I
FIG. 2. Theoretical curves showing the relationship between the fractional activity “a” and the negative logarithm of the substrate concentration as calculated from Eq. (4) assuming y = 0.1. K,, = 1O-4 111in all cases; Kd? = 1 X lo-* IIf, 4 X 10-d di, 1 X 10-J &I, 4 X 1O-3 iW, 1 X lOma LIT, and 4 X 10e2 111for the curves in order from left to right. substrate. The superficial similarity between such curves and those given by Mendel and Rudney (12) is apparent from Fig. 1. The results of Alles and Hawes (l), Mendel and Rudney (12), and Augustinsson (7) show that the a vs. log S curves are somewhat asymmetrical in most cases with the ChE’s. One explanation of this asymmetry might be that the complex ES, is not completely inactive. This does not seem unreasonable in view of the fact that a substrate such as methyl acetate is hydrolyzed slowly by the true ChE (9) although it
476
DAVID K. MYERS
would combine only with the ester grouping and not with the anionic grouping nor with other substituent groupings which would combine with the choline moiety of the Ach molecule. Similarly we might suppose that an Ach molecule which is attached to the true ChE only by the acetate moiety might also be subject to slow hydrolysis. We could postulate that the ratio of activity of ES2 to activity of ES is equal to a certain fraction
y. Then the total fractional
activity
a should be equal to 7
+ y T.
E’&=E-ES-E,=E-ES(l+%) and substituting
the value of ES/E from Eq. 2, then
ES2 -= E
Thus, for the circumstances
a=T+y+
sz Se + K.,S + KJL,’
outlined
above,
K,,S + YS= P f K,,S + K.,K.,’
(4)
If we assume that y = 0.1, for example, and construct a series of curves analogous to those shown in Fig. 1, we obtain the asymmetric bell-shaped curves of Fig. 2. As the substrate concentration is increased to very high values, a point should be reached at which the enzyme activity becomes minimal; further increases in the substrate concentration should not affect the activity significantly. It did not always prove possible to determine whether this criterion was fulfilled in the case of excess substrate inhibition of the ChE’s but in many cases it was essential to use Eq. (4) to obtain a theoretical curve which would fit the experimental results.
EXPERIMENTAL
RESULTS
The general type of results obtained by Mendel and Rudney (12) with the true ChE of mouse brain was readily confirmed in the present investigation with the true ChE of human erythrocytes. This is illustrated by the protocol given in Table I. It can be seen that there is very little change in the true ChE activity when the concentration of Ach is increased from 3 X lo4 M to 1O-3 M in a 0.025 M sodium bicarbonate medium, which would indicate that the optimal concentration of Ach lies in this range. In the presence of 0.5 M sodium chloride, the true ChE activity at both concentrations of Ach is considerably lower than in the presence of 0.025 M bicarbonate alone; further, the true ChE activity increases markedly as the concentration of Ach is increased from 3 X 10” M to 10m3M indicating that the optimum must lie at a considerably higher range of concentrations under these conditions. The true ChE activity of the erythrocytes was similarly determined with a series of Ach concentrations ranging from 3 X lo4 M (5 mg.-%)
HYDROLYSIS
OF
ACETYLCHOLINE
477
to 0.2 M (3.3%) with an increase in concentration of approximately 1.5 times at each step of the series. The results were plotted graphically against the logarithm of the substrate concentration and the best theoretical curve was fitted to these points by a series of trials with decreasing error. It was found that the results obtained with a 0.025 -U bicarbonate medium could not be fitted by any of the symmetrical bell-shaped curves derived from Eq. (2). Different forms of the asymmetrical curves given by Eq. (4) were then tried; the theoretical curves for which y = 0.1 proved to give a relatively good fit to the experimental results. This value of y was also assumed for the other experimental curves where the value of y could not be accurately determined (e.g., in the presence of 0.2 and 0.5 M NaCI). The experimental results correspond fairly well to these curves in all cases but would also correspond to theoretical curves in which y < 0.1. However, the values of V,., and K,, would hardly be affected by the assumption of a smaller value for y in the presence of higher concentrations of sodium chloride, and the error introduced in the estimations of K,, would be relatively small. The general trend of the results will not be affected by this assumption. The curves obtained are presented in Fig. 3; it can be seen that these curves resemble closely the experimental curves given by Mendel and Rudney (12). Most of the values of K,, and K,, can be determined with some accuracy from the experimental results except in the case where a 0.025 211bicarbonate medium is used. The lowest concentration of Ach at which the activity can be tested is limited to about 3 X lo4 M by this technique, and the true ChE activity in a 0.025 A1 bicarbonate medium shows very little change with changes in the Ach concentration between 3 X 10m4J1 and 2 X 1O-3 M. However, the average of several series of experiments indicated that the activity at 6-8 X 1O-4 ikl was slightly higher than the activities at either lower or higher concentrations of Ach. Thus a theoretical curve was drawn with an optimum at approximately 7 X lop4 AT but this value must be regarded as somewhat indefinite. The shift in the optimum concentration after addition of as little as 0.02 M NaCl or 0.005 M CaCl* was quite definite nevertheless. The addition of increasing concentrations of sodium chloride to the medium caused a progressive increase in the values of both K,, and K,, (Fig. 3). The value of K,, was increased tenfold by the addition of 0.5 M sodium chloride to a 0.025 M bicarbonate medium, whereas the
478
DAVID
K.
MYERS
I GO
I a.0
.L -9 0
80
40 20
-logs FIG. 3. Experimental results obtained for the hydrolysis of Ach by the true ChE of human erythrocytes in the presence of various concentrations of NaCl increasing in order from left to right. The ordinate gives the enzyme activity as 4. COJO.1 ml. erythrocyte hemolyzate/20 min., the abscissa gives the negative logarithm of the molar concentration of Ach. The theoretical curves were calculated from Eq. (4) assuming y = 0.1; the best theoretical curves were obtained using the following values:
0.55 1.10 200 0.78 0.021 1.10 1.21 110 1.15 0.05 1.9 1.35 70 1.60 0.10’ 2.6 1.55 60 2.05 0.20 3.7 1.8 50 2.70 0.50 5.5 2.2 40 3.65 1 To avoid unnecessary confusion on the graph, these two curves in Fig. 3.
140 157 146 169 156 190 168 208 175 221 171 221 were not included
HYDROLYSIS
OF
ACETYLCHOLINE
479
value of K,, was increased only by twofold so that the ratio of K,, to K,, becomes progressively smaller. The optimum concentration of Ach is also shifted to higher values as shown by Mendel and Rudney (12). The absolute rate of hydrolysis at the new optimum level was increased
2oo I-
Fro. 4. Experimental results obtained for the hydrolysis of Ach by the true ChE of human erythrocytes in the presence of 0.025 M sodium bicarbonate alone (left) and with 0.005 M calcium chloride added (right). The theoretical curve for activity in the 0.025 M bicarbonate medium is the same as that given in Fig. 3; the curve for the activity in the presence of CaClz was calculated on the same basis using the following values: K., = 2.75 X 1O-4 M Ach, K,, = 1.10 X lO-e M Ach, K.,/K., = 40, f&t = 1.9 X 10m3III Ach, V,,t = 186 ~1. COJ20 mm., Vu, = 241 ~1. CO*/20 mm.
227c by the addition of 0.5 M NaCl, but the theoretical maximum was increased even more (41%). This is because the progressive decrease in the ratio K,,/K,, tends to mask this activation to some extent. The activation of the true ChE activity toward Ach is even more
480
DAVID
K.
MYERS
apparent with a bivalent salt such as calcium chloride. In this instance a special technique was necessary since a slow reaction liberating free CO, occurs when a bicarbonate medium containing CaClz is incubated at 37”. In the presence of erythrocyte hemolyzate the calcium ion is apparently bound by the proteins and the reaction does not occur (17). The controls for this experiment contained erythrocyte hemolyzate
200
-
180
-
IGO I40
-
120
-
100
-
8o 60
-
40
-
> -$ .-
2
FIG. 5. Experimental results obtained for the hydrolysis of Ach by the pseudo-ChE of human serum in the presence of 0.025 M sodium bicarbonate alone (left) and with 0.5 M sodium chloride added (right). The ordinate gives the enzyme activity as ~1. COP/O.1 ml. serum/20 min.; the abscissa gives the negative logarithm of the molar concentration of Ach. The theoretical curves were calculated from Eq. (1) using the following values: Medium
K.
hW.X
0.025 M NaHCO, + 0.5 M NaCl
1.38 x 10-s M Ach 2.68 x 10-S M Ach
206 ~1. CO*/20 min.
175 j4l. CO&Kl min.
HYDROLYSIS
OF
ACETYLCHOLINE
481
with 10m6M diisopropyl fluorophosphonate added to inhibit completely the true ChE activity toward Ach. The results obtained by this method are shown in Fig. 4. In contrast to the effects of NaCl, 0.005 211CaClz appeared to have very little effect upon the value of K,, even though the value of K,, was increased markedly. The optimum concentration of Ach is also shifted to higher levels, but the most striking effect of the small concentration of CaClz is the strong activation of the maximal activity of true ChE toward Ach. It is usually assumed that salts have little effect upon the hydrolysis of Ach by pseudo-ChE but it was possible to demonstrate a slight shift in the affinity constant in the present investigation when high concentrations of sodium chloride were employed. This agrees with previous experiments in which it was shown that the presence of sodium and potassium chloride does inhibit the activity of pseudo-ChE towards suboptimal concentrations of Ach (9,18). Determinations of pseudoChE activity with a series of different concentrations of Ach showed that the affinity of pseudo-ChE for Ach was significantly decreased (1.9 times) by the addition of 0.5 M NaCl to the medium (Fig. 5), although the decrease in affinity (or increase in K,) is not nearly as striking as in the case of the true ChE. The experimental results could be fitted quite well by a simple equation of form 1. It appeared that the maximal activity wasPlightly lower in the presence of salt than in its absence, an effect which is not dependent on the increase in K,. This would indicate that the salt acts not only as a simple reversible competitive inhibitor (K, increased, V,, unchanged) but also as a noncompetitive inhibitor (K, unchanged, V’,,, decreased). DISCUSSION
It has been shown that the maximal activity of the true ChE toward Ach is considerably potentiated by the presence of salts, and especially by a bivalent salt such as calcium chloride. However there has not been any conclusive evidence to indicate that the presence of salts is essential for the activity of either the true ChE or the pseudoChE’s of mammalian tissue. There is very little loss in activity when the true ChE of hemolyzed erythrocytes or the pseudo-ChE of serum is subjected to ordinary dialysis against distilled water for 24 hr. as in the present and previous (8,9) investigations. It is essential that the pH should be in the neighborhood of 7, and it is preferable that the dialysis
482
DAVID
K.
MYERS
should be carried out at. a low temperature (3°C.) t,o prevent inactivation. This might appear to contrast with the results of Nachmansohn et al. (19,ZO) who found that the true ChE of the electric organ of Torpedo vulgaris was inactivated to a large extent, by the process of dialysis. However, these investigators concluded at the time that much of the inactivation was caused by the increased sensitivity of the dialyzed enzyme to cont,aminating traces of heavy metals and of copper in particular. They showed that the true ChE activity was dependent upon t’he presence of free sulfhydryl groups which were readily inactivated by the metallic impurities. It was concluded in a later investigation that the addition of bivalent cations (e.g., Gaff, Mg++, &In++) gave a reactivation of the dialyzed enzyme which was independent of the reactivation of sulfhydryl groups (21). It seems possible that this might be due in part to a shift in the optimum concentration of Ach to higher values in the presence of salt. When this work (21) was done, it was not known that these were two types of ChE and that the true ChE exhibits the phenomenon of excess substrate inhibition by Ach. The concentration of Ach employed was fairly high (0.016 M) and the enzyme would be strongly inhibited by the excess substrate since the activity-substrate concentration relationship with this enzyme preparation in the presence of 0.025 M bicarbonate is very similar to t,hat shown by the true ChE’s from mammalian tissues.2 Addition of salts to the medium would lead to a shift in the optimum concentration of Ach to much higher levels, and this in itself would appear to result in a large activation of the true ChE activity at a high concentration of Ach. This may not be a complete explanation of the above-mentioned results (Zl), but a full interpretation of the results with this enzyme preparation is impossible without a full picture of the enzyme activity at a series of Ach concentrations (22). It does seem possible by analogy with other enzymes that the presence of a very small amount of a bivalent cation may be essential for the activity of the ChE’s (23), but the experimental evidence has not been convincing. Bivalent salts do cause an appreciable activation of the pseudo-ChE (18) and true ChE activity, but prolonged dialysis has very little effect upon the activity of a crude or a highly purified preparation of pseudo-ChE (23, 24, 25). Moreover the results described ppOl~~~~~, B., AND RUDNEY, H., unpublished.
HYDROLYSIS
OF
ACETYLCHOLINE
483
above show that a simple salt such as sodium chloride also possesses the property of activating the true ChE albeit to a much smaller extent than calcium chloride. It has been suggested that the bivalent cation may be very strongly held by the ChE itself under the conditions of the dialysis (23) since the ChE activity is partially inhibited by substances which form insoluble salts with Ca++ (e.g., fluoride, oxalate, citrate, pyrophosphate) when present in high concentrat’ions (23). This latter fact would seem to substantiate the hypothesis that Ca++ or a similar ion does form an essential part of the ChE molecule if it is assumed that the action of these compounds upon ChE is due solely to the removal of bivalent cations from the solution. The reasons for the failure of some other investigators to confirm the shift in optimal Ach concentration for true ChE caused by the addition of salts have been discussed previously. The results obtained in the present investigation are in good agreement with the results of Alles and Hawes (1) and Mendel and Rudney (12). L411esand Hawes (1) used a t,itrimetric technique and were able to determine the true ChE activity accurately at much lower concentrations of Ach than is possible with the manometric technique used above. Theoretical equations of the same type as used above were fitted to their experimental data to give the curves illustrated in Fig. 6. It would appear from these curves that K,, = 2 X lop6 211Ach and K,, = 8 X 1O-3 ill -4ch for the true ChE of erythrocytes in the presence of 0.0059 111 NaCl. These values fit very well into the series of values obtained in the present investigation and offer substantial confirmation of the above results. The data of Mendel and Rudney (12) were concerned especially with the optimum concentration of Ach in the presence of various concentrations of salt and do not cover a sufficiently wide range of Ach concentrations to allow an accurate determination of K,, and K,, from the experimental data. However, a comparison of the optimal concentration of Ach and the activity at each optimum level (V,,,) shows a close agreement between their results and those obtained above (Table II). The one point of significant discrepancy is with regard to the optimal concentration of Ach in the presence of 0.025 JU sodium bicarbonate alone, the values obtained being 2.5 X lo-’ -V ,4ch and 7.8 X 1O-4 iW ilch in the two investigations, respectively. However, the true ChE’s employed were from very different sources (mouse brain and human erythrocytes). Moreover, the activity-substrate concentration curves
484
DAVID
K.
MYERS
are relatively flat in this concentration range so that the precise value of the optimum concentration cannot be accurately determined. The results obtained in the present investigation indicate that sodium chloride has two distinct actions upon the true ChE activity toward Ach. In the first place, as suggested previously, it acts as a reversible competitive inhibitor and causes an apparent increase in the
I.0
0.8 > 4-J ..-> d 0.G 0.4
0.2
results obtained by Alles and Hawes (1) and the corresponding FIG. 6. Experimenta theoretical curves used in the present investigation to fit these results. The ordinate is true ChE activity towards Ach expressed as milliliters of 0.02 N NaOH/20 mm.; the abscissa is the negative logarithm of the molar concentration of Ach (calculated from the experimental curves of Alles and Hawes where the concentration was expressed as miUimoles of Ach per 25 ml. of solution). The theoretical curves were cdculated from Eq. (4) assuming y = 0.1 and using the following vaiues:
0.0059 0.145
0.20 1.0
0.80
0.5
1.0
LO
0.81 1.32
values of K+ and of K,,, especially of the former. This leads to an increase in the optimum concentration of Ach. The second effect is an activation of the true ChE. This is probably similar to the effects of salt as previously described for the hydrolysis of Ach by the true ChE of Planaria (26) and for the hydrolysis of non-choline esters by the true ChE of human erythrocytes (9). It has been suggested previously that
HYDROLYSIS
OF
485
ACETYLCHOLINE
the activation by salts may be due to an interaction with the ester grouping of the true ChE active center (9). It is not clear how this could be correlated with the supposition that Ca++ (or a similar cation) forms an essential part of the active center, or with the theory (11) that the ester grouping of the active center contains a latent basic group and a latent acid group in its active condition. Like most other proteins, the true ChE molecule as a whole (or, at least, the particulate matter with which the true ChE is associated) possesses a negative charge at physiological pH (2T), and it is probable that this charge would be affected not only by alterations in pH but also by alterations in the concentration of salts in the medium. It is very difficult to see how this latter fact alone could explain the selective effects of salts upon ChE activity and inhibition [cf. Refs. (2,8,9,12,21,24,28)-J; it seems necessary to assume that the salts also have a specific effect upon the enzyme active center. TABLE II Comparisonof the Data of MeruZel and Rudney (1.2) and Those of the Present Znvestigatim Illustrating the Effect of Salts upon the Hydrolysis of Ach by True ChE Data of Made1
and R~;~neney(12)
Medium
0.025 M +0.04 $0.08 +0.16
NaHCOs M KC1 M KC1 M KC1
V0pt
0.25 1.5 2.0 3.0
100 118 122 125
Data from present investigation &t Medium
0.025 M +0.05 +O.lO +0.20
NaHCOs M NaCl M NaCl M NaCl
0.78 1.60 2.05. 2.70
100 111 120 125
The evidence in favor of a dipositional active center for the true ChE has been critically reviewed recently by Whittaker (29). Three groups of investigators working independently from three different approaches have produced evidence for the existence of an anionic position in the enzyme active center (8,10,30). It would be expected that the anionic position has the effect of increasing the affinity of the true ChE for Ach and analogous esters and of facilitating the hydrolysis of the ester linkage by the esterasic site (29), or “ester grouping.” In so far as the salt acts as a reversible competitive inhibitor of true ChE, we could postulate as in the previous investigations (8,9) that the cation competes with Ach for the anionic position in the ChE active center. The addition of sodium chloride to the medium decreased the affinity
486
DAVID K. MYERS
of Ach both for the true ChE and for the pseudo-ChE. It would follow that the active center of the pseudo-ChE as well as that of the true ChE is probably dipositional in nature. However the combination of Ach with the pseudo-ChE does seem to be much less dependent upon the cationic ammonium group of Ach than is the case with the true ChE. The experimental results obtained previously with t#he true ChE (8) show that the addition of 0.475 M NaCl to a bicarbonate medium decreased the affinity for eserine by sixfold (-log 160 decreased by 0.78). The above results indicate that 0.5 M NaCl decreases the affinity of the true ChE for Ach by even more, i.e., tenfold (K,, increased ten times). For the pseudo-ChE, on the other hand, the addition of 0.5 M NaCl resulted in a change of 5.2 times (antilog 0.72) with prostigmine (9), 3.8 times (antilog 0.58) with eserine (9), and 1.9 times with Ach. This could be interpreted as meaning that the affinity of the pseudo-ChE for Ach is much less dependent upon the anionic position of the enzyme active center than is the case with eserine or prostigmine. The reason for the difference between Ach and prostigmine is not apparent, but the conclusion in itself does agree with the results of other workers who have shown that the binding of Ach by pseudo-ChE is mainly dependent upon the ester structure of the Ach molecule (30,31). ACKNOWLEDGMENTS The author is indebted to Dr. B. Mendel for his criticisms and suggestions, and to Misses M.. de Jonge and E. Simons for their technical assistance. SUMMARY 1. In confirmation of the results of Mendel and Rudney, it has been shown that the optimum acetylcholine concentration for true cholinesterase is shifted to progressively higher levels by the addition of increasing concentrations of salt to the medium. 2. The theoretical equations for Murray and Haldane have been applied to the resulting curves between enzyme activity and substrate concentration. It has been found that these equations will not fit the experimental results unless the assumption is made that the “inactive” complex ES, is subject to slow hydrolysis. The relative activity of the ES2 complex would appear to be about one-tenth of the activity of the ES complex between enzyme (E) and substrate (S). 3. The addition of simple salts (e.g., NaCI, KCl) decreases the affinity of true cholinesterase for acetylcholine. The salts have a further
HYDROLYSIS
OF ACETYLCHOLINE
487
effect in potentiating the hydrolytic activity of true cholinesterase toward acetylcholine; this activation is especially marked with a bivalent salt such as calcium chloride. The significance of these results has been discussed in relation to the theory of a dipositional enzyme active center. 4. The results support the supposition that the active center of the pseudocholinesterase is also dipositional in nature but the affinity of acetylcholine for pseudocholinesterase appears to be dependent mainly on the ester structure of the acetylcholine molecule. REFERENCES 1. ALLES, G. A., AND HAWES, R. C., J. Biol. Ch.em. 133, 375 (1940). 2. MENDEL, B., AND RUDNEY, H., Biochem. J. 37, 53 (!943). 3. ZELLER, E. A., AND BISSEGGER, -4., Helv. Ch.im. Acta 26, 1619 (1943). 4. MENDEL, B., MUNDELL, D. B., AND RUDNEY, H., Biochem. J. 37, 473 (1943). 5. MURRAY, D. R. P., Biochem. J. 24, 1890 (1930). 6. HALDANE, J. B. S., Enzymes. Longmans, London, 1930. 7. AUGUSTINSSON, K.-B., Beta Physiol. &and. Suppl. 15, 52 (1948). 8. MYERS, D. K., Arch. Biochem. 27, 341 (1950). 9. MYERS, D. K., Arch. Biochem. Biophys. 31, 29 (1951). 10. WILSON, I. B., AND BERGMANN, F., J. Biol. Chem. 185, 479 (1950). 11. WILSON, I. B., AND BERGMANN, F., J. Biol. Chem. 186, 683 (1950). 12. MENDEL, B., .&ND RODNEY, H., &%nce 102, 616 (1945). 13. NACAMANSOHN, D., AND ROTHENBERG, hI. A., J. Biol. Chem. 158, 653 (1945). 14. AUGUSTINSSON, K.-B., AND NACHMANSOHN, D., Science 110, 98 (1949). 15. AUGUSTINSSON, K.-B., The Enzymes, Vol. 1, p. 443. Academic Press, New York,
16. 17.
18. 19.
20. 21. 22.
23. 24.
25. 26. 27. 28. 29. 30. 31.
1950.
MICHAELIS, L., AND MENTEN, M. L., Biochem. 2. 49, 333 (1913). See KREBS, H. -4., 2. physiol. Chem. 217, 191 (1933); KREBS, H. A., AND EGGLESTON, L. V., Biochem. J. 34, 442 (19-U)). MENDEL, B., hIUNDELL, D. B., AND STRELITZ, F., Nature 144, 479 (1939). NACKMANSOHN, D., AND LEDERER, E., Compt. rend. sot. biol. 130, 321 (1939). NACHMANSOHN, D., AND LEDERER, E., Bull. sot. chim. biol. 21, 797 (1939). NACA~TANSOHN, D., Nature 145, 513 (1940). WYNNE, A. M., Ann. Rev. Biochem. 15, 35 (1946). MASSART, L., AND DUFAIT, R., Enzymologia 6, 282 (1939). MENDEL, B., MUNDELL, D. B., AND STRELITZ, F., Nature 145, 822 (1940). STRELITZ, F., Biochem. J. 38, 86 (1944). HAWKINS, R. D., AND hIENDEL, B., J. Cellular Comp. Physiol. 27, 69 (1946). AUGU~TINSSON, K.-B., Arkiv Kemi Mineral. Gwl. 18A, No. 24 (1944). MEER, C. VAN DER, Chem. It’eekblad, 48, 118 (1952). WHITTAKER, V. P., Physiol. Revs. 31, 312 (1951). ADA~G, D. H., AND WHITTAKER, V. P., Biochem. et Btiphys. Acta 4, 543 (1950). ELEY, D. D., AND STONE, G. S., Biochem. J. 49, XXX (1951).