of · was not affected by dfp,5 the inhibition of the wheat enzyme would offer additional evidence...

THE REVERSIBLE INHIBITION OF ACETYLESTERASE BY DIISOPROPYL FLUOROPHOSPHATE AND TETRAETHYL

PYROPHOSPHATE*

BY EUGENE F. JANSEN, M.-D. FELLOWS NUTTING, AND A. K. BALLS

(From the Enzyme Research Laboratory, Bureau of Agricultural and Industrial Chem- istry, Agricultural Research Administration, United States Department of

Agriculture, Albany, California)

(Received for publication, June 2, 1948)

Diisopropyl fluorophosphate (DFP) has been shown to be a remarkably potent inhibitor of the enzymatic hydrolysis of acetylcholine (1). As a result of a wide survey (2), inhibition by dialkyl fluorophosphates appears to be specific for certain esterases and lipases (kidney acid phosphatase was found to be inhibited by relatively high concentrations). Since the cholinesterase of mammalian brain and nerve tissue is narrowly specific for acetylcholine and acetyl-@methylcholine (3), it is often called “true” cholinesterase. Other esterases of wider specificity (those hydrolyzing tributyrin, for example) also hydrolyze acetylcholine, but are frequently referred to as “pseudo” cholinesterases.

While studying the inhibition of cholinesterase by DFP in vitro and in z&o, Mazur and Bodansky (4) found that different tissue cholinesterases show differing degrees of sensitivity to DFP. These results have been interpreted by Hawkins and Mendel (5) and by us (6) as indicating that pseudocholinesterase is about 100 times as sensitive to the inhibitory action of DFP as is cholinesterase. From a correlation of toxicity with inhibition of brain cholinesterase, Nachmansohn and Feld (7) have concluded that the toxicity of DFP is very probably due to its action on “true” cholinesterase. Since dialysis or dilution of cholinesterase-DFP mixtures or of tissue cholinesterase from animals poisoned with DFP did not result in any increase in cholinesterase, the DFP inhibition of brain cholinesterase is not to be considered readily reversible (4). However, Nachmansohn et cd. (8) have found that over a relatively short period of time the inhibition by DFP can be reversed by dilution, the extent of the reversal being dependent on temperature, time, and DFP concentration. Eserine, which inhibits cholinesterase reversibly, was found to protect the enzyme against irreversible DFP inhibition (9). Mazur (10) also demonstrated an enzyme in animal tissues capable of hydrolyzing the phosphorus-fluorine bond of alkyl fluorophosphates, so that there exists in vivo concurrent inhibition of cholinesterase and detoxification. Hexaethyl tetraphosphate (HETP) exerts a

* Enzyme Research Laboratory Contribution No. 113.

976

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976 BCETYLESTERASE INHIBITION

strong inhibitory effect similar to that of DFP on mammalian and insect cholinesterase in vitro and in viva (11). Even smaller concentmtions of HETP than of DFP were needed to cause the same inhibition.

The effect of DFP and HETP on enzymes of plant origin was studied to determine whet.her any of the plant enzymes jvere inhibited, particularly since HETP and like substances are beginning to find use as insect,icides. A preliminary report of the results has been published (6). Of t’he enzymes studied only acetylesterase (12) was found to be inhibited by DFP, HETP, or t’etraethyl pyrophosphat,e (TEP) (t,he chief active con&uentJ of HETP).

Acetylesterase occurs in cit.rus fruit and many other higher plants and fungi.’ It hydrolyzes best the esters of acetic acid, including b0t.h triacetin and acetylcholine. However, very high concentrations of acetylcholine were needed to realize maximum activity, and eserine was without, effect, on this enzyme.

A study has been made of the conditions necessary for t,he inhibition of (plant) acetylesterase, and of the kinetics of that inhibition. It was further observed that, xvhen acetylesterase preparations were inhibited by TEP or HETP, they spontaneously t’hough slowly regenerated i,n vitro. Purified preparations of another enzyme from orange flavedo (pectinesterase) frequently carried some material which accelerat.ed this regeneration. On t,he other hand, enzyme inhibited by DFP has not been observed to regenerate in vitro, though evidence was obtained that, when a whole orange was gassed with DFP, marked inhibition of acetylesterase and also some subsequent regeneration thereof occurred in the intact fruit.

EXPERIMENTAL

Methods and Materials

Enzymes and Assay Methods-The following enzymes and assay methods were employed: Arlington jack bean urease was assayed according to the aeration-titration method of Van Slyke and Archibald (13), papain salt paste (14) according to the milk-clotting method of Balls and Hoover (15), crystalline P-amylase (16) according to the Schwimmer modification (17) of the Kneen and Sandstedt method (18), crude pectinesterase concentrate according to the method of continuous titration at constant pH (19), and purified acetylesterase according to a similar continuous titration method previously reported with diacetin or triacetin as a substrate (12), or by a calorimetric method recently developed with o-nitrophenyl acet’ate as substrate.

The calorimetric method for acetylesterase permitted the use of a tenth as much enzyme as was needed for the titrimetric methcd. It consisted

1 MacDonnell, L. R., Jang, R., Jansen,, E. F., and Lineweaver, H., in preparation.


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E. F. JANSEN, M.-D. F. XUTTING, A. K. BALLS 977

in measuring the rate of formation of o-nitrophenol at pH 6.5 and 25’, with a No. 42 filter in a Klett-Summerson calorimeter with the test-tube adapter in place. The substrate was dissolved in alcohol and added to the enzyme solution in 0.1 al phosphate buffer at pH 6.5 (below the surface) to give a final concentration of 2 per cent alcohol and 0.003 M o-nitrophenyl acetate. The color formed was measured as a function of time. The rate of formation under t.hese conditions was linear and was directly proportional to the amount of enzyme. A standard curve related color to m&f of o-nitrophenol. The activities were expressed in millimoles of o-nitrophenol formed per minute per aliquot of enzyme. These activities agreed with those deter- mined with triacet,in as a substrate to within 10 per cent. The small sub- skate concentration needed to show the full activity of the enzyme present is noteworthy (concentrations of o-nitrophenyl acetate greater than 0.003 M gave t.he same specific activities). This concentration is less than 0.01 of that necessary for full activity with the acetins as substrates (12). Thence acetylesterase has a much greater affinity for phenyl acetates than for aliphat.ic acetates.

I;nhibilors-The HEW” was a commercial product known t.o be a mixture, t,he DFl? was analytically pure, the TEP* was 95 per cent pure, and no analytical figures were available for the ethyl metaphosphate (EMP).4 Stock solutions of the inhibitors were made to be 0.01 to 0.02 III in anhy- drous isopropanol. These solutions kept for several months in the refrig- erator with no decrease in inhibitory power. Aliquots of these solutions sufficient to give the desired concentration of inhibitor were added to an enzyme solution and incubated at room temperature (25” f 1”) for 20 minutes (unless otherwise specified) prior to assay. It is shown later that an incubation period is necessary. As a control, pure isopropanol was added in a corresponding amount to a similar enzyme solution in order to ascertain whether any of the inhibition observed might be due to the isopropanol. In no case did the amount of isopropanol used produce any inhibition over the time of the experiment. The term pK is used to denote the negat,ive logarithm of the concentration of inhibitor required to produce 50 per cent inhibit,ion under the described conditions.

Resdts

EJect of DPP O)L Several P&t Enxymes--The effect of DFP on papain, pect,inest)erase, urease, crystalline P-amyla.se, and citrus acetylesterase is

z Obtained through tjhe courtesy of the hIonsanto Chemical Compauy, St. Louis, Missouri.

a Obtained from the Medical Division, Army Chemical Center at Edgewood, Maryland, through the courtesy of Captain James A. Campbell.

4 Kindly supplied by Dr. Howard Adler of the Victor Chemical Works, Chicago, Illinois.


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978 ACETYLESTERASE INHIBITION

given in Table I. It can be seen that the arbitrary concentration of DFP employed (0.001 M) had little or no effect on any of the enzymes tried except acetylesterase and possibly papain. (The pH had no effect on DFP inhibition, as will be seen below; therefore, it was not possible that the failure of DFP to inhibit was due to pH differences.) Increasing the concentration of DFP in papain solution 3-fold caused an inhibition of 25 per cent. This inhibition was not dependent upon time, since 30 and 90 minute incubation periods caused the same inhibition, contrary to the effect observed in the inhibition of acetylesterase (see beloJv). Since t,he concentrations of DFP necessary for papain inhibition were so large, the effect was not pursued any further. Urease and papain depend on -SH groups for activity. Since DFP failed to cause a marked inhibition of these enzymes,

TABLE I

Effect nj 0.001 M DFP on Seoeral Plant Enzymes __-

EIl.Q%e / PB i Incubation time* Per cent inhibition --

min.

Papain?. 4.7 30 9 ‘I .,...,.................,.,. 4.7 180 9

Pectinesterase..................... 5.6 30 0 Urease............................ 7.0 90 0 /3-Amylase. 5.9 60 2 Acetylesterase (citrus). 6.7 ‘7 84

I‘ ‘I 6.7 43 / g6-_

* The time between the addition of the DFP to the enzyme and its assay. f No cyanide activation was used on the papain.

it is apparent that the mode of inhibition by DFP is not through reaction with -SH groups. It is of interest that pectinesterase is an esterase un- affected by DFP.

Effect of Concentration of DFP on Acetylesieyase-In order to determine the concentration of DFP necessary to cause 50 per cent inhibition of acetylesterase, it was necessary to select an arbitrary t,ime of reaction between the enzyme and the inhibitor. For this purpose a 20 minute incubation period was chosen. The suggestion had been made previously (12) that wheat ‘Llipase” (20) is in reality acetylesterase. Since lipase (pancreatic) was not affected by DFP,5 the inhibition of the wheat enzyme would offer additional evidence of the identity of this enzyme with acetylesterase. A concentrate of the wheat enzyme was made by extraction of 100 gm. of wheat germ overnight with 1 liter of 2.5 N sodium chloride at pH 7.0. The filtered extract was made 0.7 saturated with ammonium sulfate and the

6 Unpublished results.


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E. F. JANSEN, M.-D. F. NUTTING, A. K. BALLS 979

precipitate was filtered off and taken up in 75 ml. of water and dialyzed in the cold. The result.ing solution had an activity of [AE u.] ml. = 0.002. Aliquots of this solution as well as those of citrus acetylesterase were sub- jected to DFP. From Table II it is apparent that the concentration of DFP necessary for 50 per cent inhibition under the conditions of the experiment was approximately 5 X 1O-5 M (pK = 4.3) for both preparations. This concentration is of the same order as that necessary for 50 per cent inhibition of true cholinesterase (4, 21). The fact that the same concentration of DFP caused a similar inhibition of the enzymes from both sources is additional evidence that the wheat enzyme is in fact acetylesterase. That the inhibition by DFP was not due to its hydrolysis to fluoride ion was shown by the failure to obtain inhibition of acetylesterase by incubation with 0.001 M NaF.

Citrus flavedo press-juice contains considerable acetylesterase (12). The concentration of DFP necessary to cause 50 per cent inhibition in a similar

TABLE II Effect of DFP Concentration on Acetylesterase

DFP concentration X 104 Per cent inhibition’

Citrus acetylesterase I Wheat acetylesterase

5 86 1 72 0.5 56

* After 20 minutes prior exposure of the enzyme to DFP.

91 75 65

incubation period was found to be approximately 10 times as much as was needed for the partially purified acctylesterase used above. On purification of the acetylesterase (12) this difference became negligible. The explana- tion of these observations is still obscure, but obviously may be connected with the observations on regeneration of the inhibit,ed enzyme.

Kinetics of DFP Inhibition-A study was made of the rate of inhibition of citrus acetylesterase by DFP at 25”. The reaction was found t’o be bimolecular over the concentration range studied, since the product of the time necessary to cause 50 per cent inhibition multiplied by the inhibitor concentration gave a constant (Table III), except for t.he lowest concentration studied. Furthermore, a plot of the reciprocal of residual activity against time gave a linear function for more than half of the reaction. This may be considered as additional evidence that the inhibiting reaction is bimolecular. The DFP concentrations used were all above those with which Nachmansohn et al. (8) were able to obtain reversible inhibition, SO that it seems unlikely that the change from reversible to irreversible inhibition


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influenced these results. Hence in order to define DFP inhibition with acetylest’erase the time as well as the concentration must be considered. The inhibition of cholinesterase has likewise been shown to be a reaction dependent upon time (8, 22).

Non-Dependence oj DFP Inhibition 01% pPi--Aliquots of wheat acetylesterase concentrate were adjusted to pH 4.9, 6.2, and 7.4, and incubated with 5 X 10e5 M DFP. The amount of inhibition observed was 50, 56, and 53 per cent, respectively. The small differences are not considered to be significant,. Similarly, acetylesterase in citrus press-juice at pH 5.4 and 6.1 was inhibited under like condit8ions to the extent of 17 and 20 per cent, respectively. Hence over the range of pH studied the reaction of DFP with acetylesterase was not dependent upon pH.

Efect of Eserine and Substrate on DFP Inhibition-The fact that eserine did not inhibit the hydrolysis of acetylcholine bromide by citrus acetyl-

TABLE III DE’P Inhibitiotl, a Bimolecular lieaction

I DFP concentration X 10s ’ Time of 50 per cent inhibition (ta) ’

_-- -1 1’, X concentration X 104

------- ---.- __._, ___-.-.~---- __- min.

1 83 8.3

,;I 21 12 / 10 12 100 1 I 10

___~-~-- -.-.. -___ _-..-

esterase has previously been demonstrated (12). Wheat acetylesterase was likewise found to hydrolyze acetylcholine bromide; however, at a concentration of 0.9 M the relative rate of hydrolysis (on %he basis of triacetin activity as unity) was only 60 per cent, of that, of cit,rus acetylesterase. In- cubation of wheat acetylesterase at 25” for 90 minutes in 1 X lo-* M eserine failed to show any inhibition of acetylcholine hydrolysis on subsequent assay. Moreover, t,he hydrolysis of acetylcholine by the wheat enzyme in t’he presence of 1 X lo-” M eserine proceeded at the same rate as in 6he absence of eserine. Treatment with 5 X low5 M DFP of wheat acetylesterase, which had already been incubated with 1 X 10e4 M eserine for 90 minut.es, caused t,he same inhibition as was observed in t,he absence of eserine. Therefore, unlike cholinesterase, acetylest’erase was not inhibited by eserine and, probably for the same reason, the eserine did not protect acetylesterase from DFP inhibition. Conversely, Webb (2) found that the enzymes other than cholinesterase which were inhibited by DFP were sensitive to eserine, although there was no direct correlation between the sensitivity to DFP and eserine.


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In the presence of substrate the inhibition of acetylesterase by DFP is greatly retarded; i.e., in t,he presence of 5 per cent triacetin, 4-O minutes were needed to reduce the activity of the wheat enzyme to one-half by the action of 1 X 10W3 M DFP, whereas in the absence of the triacetin this degree of inhibition would have occurred in 1 minute. The t,ime needed to reduce the activity to one-half in the presence of substrate was approximately t,he same at lower conccnt’rat’ions of DFP (1 and 5 X lo+ M). It is probable, therefore, that the point of attack of t,he DFP involves the point of a,ttachment of the enzyme to the substrat’e.

Ebfeet o,f fZE”1’1’, TEE’, and EMP on dcetylesteruse-I’iETP, a mixture of ethyl phosphates, was found to inhibit acet,ylesterase (Table IV) in even smaller concentrations than n-cre needed wit’h DFP (Table II) ; approximately one-fift#ieth to one-twenty-fifth as much caused 50 per cent inhi-

Eflect of HE’I’P, TEP, und E’MP on Acetylesterase

Enzyme source Inhibitor I

’ Inhibitor : Per cent concentration I inhibition*

Wheat Hh:Tl “ “ l x lo--” /

57

/ 1 x lo- -6

Citrus, purified.. _. _. j “ ’ 2 x 10-6 / 43

50 ‘I “ / T l!: 1’ 1 x lo-” / 46 “ iL . . . . . . . . . / ESII’ I 1 x lo--” / 50

- ______.----.----. --~.-~- * After 30 minutes prior exposure of the enzyme to the inhihitjor.

bition in a like time. This difference is considerably greater than that observed for cholinesterase (11). The active insecticidal constituent of HETP,‘j TEP, was just as effective on a molar basis. However, EMP, the other major constituent of HETP, was one-tenth as act,ive as TEP. Since the EMP was impure and probably still contained residual TEP, the inhibition was probably due to the latter. Here again the reaction between HETP and acetylesterase was found to be bimolecular. Acetyles- terase in citrus press-juice required 10 times as much TEP to give 50 per cent inhibition as did t.he partially purified acetylesterase.

Regeneration of Acetylesterase--As cited earlier, the conclusion that cholinesterase is irreversibly inhibited by DFP is based largely on the behavior of the inhibited enzyme upon dialysis. Similar experiments with inhibited acetylesterase showed that dialysis, even after 11 days, produced no resto- ration of activity when the enzyme (from either wheat germ or orange

0 Private communication from Dr. H. 1,. Hailer, Bureau of Entomology and Plant Quarantine, United States Department of Agriculture.


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982 ACtiTYLESTERASE INHIBITION

flavedo) was inhibited with DFP.’ When it was inhibited by TEP or HETP, however, a small but definite increase was observed. (For example, a wheat germ preparation inhibited to 14 per cent of its original activity by 1 X 10-j M HETP rose to 17 per cent in 3 days; a citrus preparation, inhibited to 16 per cent of its original activity by 1 X 10V4 M TEP, rose to 30 per cent in eleven days.)

This regeneration of activity, however, was found not to depend upon dialysis, but to occur simply on standing. As shown in Table V, the re- appearance of activity was more rapid at room temperature than at 5”. However, in the long run, greater regeneration occurred at the lower tem-

TABLE v

Regeneration of TEP-Inhibited Acetylesterase

Inhibitor*

DFP TIT i

“ <‘ L‘

Added pectinesterase

preparationt

- - t t t (heated)!

4 hrs.

Per cent of original activity after

1 day 5 days 14 days

I I

20 days ____

21

7 23

2

15 5 3 5 34 45

27 54 29 29

32 34

* The inhibitor was added to the acetylesterase to give a concentration of 1 X 10-d II.

t A purified, lyophilized preparation of citrus pectinesterasel was added to the acetylesterase to give a concentration of 0.7 pectinesterase unit per ml.

$ After an incubation period of 4 hours at 25”, the reaction mixtures were stored at the respective temperatures.

0 A solution of the pectinestcrase preparation was heated to 60” for 5 minutes and cooled prior to its addition to the acetylesterase.

perature. The results were by no means regular and indicated to us the likelihood that some unidentified factor in the preparations was partici- pating in the observed return of activity. This supposition was strengthened by the observation that additions of purified citrus pectinesterase frequently (but not always) increased the rate of regeneration. A preparation of tomato pectinesterase, however, was without effect. The pectinesterase content of the preparations used did not appear to be corre- lated with their regenerative effect on inhibited (citrus) acetylesterase. Heated pectinesterase preparations were entirely inactive in this respect.

While preparations of citrus acetylesterase in aqueous solution were never

7 The dialyses were carried out in the cold with conditions under which acetylesterase was stable; i.e., dialysis against 0.1 n% sodium oxalate (12).


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observed to recover from inhibition by DFP, the enzyme was found to be regenerated in situ after oranges were gassed with DFP vapor. It has previously been shown that the major portion of acetylesterase occurs in the flavedo of orange rind (12). When oranges are evacuated in a desiccator containing a small amount of DFP, some of the chemical is volatilized and forced into the fruit when atmospherjc pressure is restored. By repeating the evacuation several times, it was found that over 95 per cent of the acetylesterase in the outer layers of the rind could be inhibited.

TABLE VI

Regeneration of DFP-Inhibited Citrus Acetylesterase, in Situ*

Per cent of untreated activity

Rind parrt After oranges stood at 25” After treatment -

1 day 3 days 4 days

Whole flavedo.. . 4 13 I‘ I‘ . . . . . . . . . . . . . . 5 40

Outer ‘(1 . . . . . . . . . . . ..__ 5 15 Inner “5 . . . . 5 26 Albedo.. . 50 28

* The acetylesterase was inhibited by evacuating the oranges in a desiccator containing 1 ml. of DFP for 1 minute, then allowing the oranges to remain at atmospheric pressure for 1 minute and repeating this cycle ten times. After the last evacuation the oranges were allowed to remain in the desiccator at atmospheric pressure for at least 0.5 hour. Washing the oranges after treatment had no effect on the inhibition.

t The rind parts were allowed to remain on the orange for the time indicated in the last column, after which they were removed and extracted as previously described (12).

$ The outer flavedo was that part of the flavedo which contained most of the oil, and was removed by grating.

9 The inner flavedo was that part of the flavedo which contained little oil but still relatively large amounts of pigment.

However, if such fruit were allowed to stand 3 to 4 days at 25”, the acetylesterase in the flavedo was found to have regenerated to the extent of 15 to 40 per cent of its original (uninhibited) value (Table VI). In the mean- time, the rind apparently suffered some autolytic changes, but the replace- ment of the esterase activity can hardly be attributed to diffusion from elsewhere in the fruit, for in the layers of tissue beneath the outer flavedo (as seen in Table VI) about the same degree of regeneration was observed. Consequently there exist some factors in citrus fruit capable of forming active acetylesterase after that enzyme has been inhibited by either DFP or TEP. Attempts to concentrate or identify such factors have so far met


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with no succc~s, but they will be continued for the sake of their importance, not only to certain problems of citrus metabolism but also to that of recovery from DFP poisoning.

DISCUSSION

Certain properties of plant acetylesterase as it occurs in fairly crude preparations from wheat germ and orange flavedo may now be compared with those of the choline&erases of animal tissue. The comparisons are summarized in Table VII. The general similarity between the plant and animal enzymes is striking. In both cases, the a,ction of the phosphate inhibitors appears, at the present writing, to be a specific one. While this similarity is backed by a great amount of experimental work, it is never- theless based on ignorance, for the possibilities are not nearly ex- hausted. At present the known differences are more profitably discussed.

TABLE VII Comparison of Acetylesterase with Cholinesterase

Property

hffinity for acetylcholine Inhibition by eserine Specificity pK of DFP inhibition ‘I “ HETP “

__-

Cholinesterase !

True j Pseudo / _ Acety*esterase

GtTill, I Less t- ;+

; past (Km = 1.6 &I)

Karron Wide / Wide 3.8-5 (20) j 6.5-7 (20) ’ 4.3 7-7.5 (10) j 7 (10) 6

-- The figures in parentheses are bibliographic references

The import,ant r&e of acetylesterase in plant metabolism can hardly be the hydrolysis of acetylcholine, and the observed similarities between this enzyme and the cholinesterases are more probably dependent upon the ester-hydrolyzing mechanism of all of them than upon any factor specific for choline. This is supported by the wider specificity of the plant enzyme. It should be noted also that not aliphatic, but aromatic, acetates are its preferred substrates. Thus the affinities (Km values) of acetylesterase for acetylcholine, triacetin, and o-nit.rophenyl acetate were 1.6 M, 0.03 M (12), and 0.001 M, respectively.

The inhibition constants (pK) for HETP are practically the same, but those for DFP differ widely. This suggests that the mode of action of HETP inhibition may be quite different from that of DFP inhibition.8

*The behavior of plant acetylesterase undoubtedly resembles that of animal pseudocholinesterase.


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There is a marked difference in the concentration of DFP required for the inhibition of the three enzymes shown in Table VII, but no difference in the concentration required of HETP. This again suggests that the mode of the two inhibitions may be different, a supposition that is borne out by the observation that the plant? enzyme, while it, recovers from both inhibitions, does so under totally different circumstances. The reappear- ance of the HETP-inhibited enzyme depends upon an unknown factor in the water-soluble preparations. The factor is evidently heat-labile and operates faster but not more extensively a.t higher temperatures. These properties are charact,eristics of an enzyme. After being inhibited by DFP, acetylesterase reappears only when in contact with the original tissue, and during a process outwardly resembling autolysis of that tissue. It has not been technically possible to inhibit citrus acetylesterase by HETP in situ apparently because of the difhcult,y of volatilizing enough of the chemical. Application of a solution t.o the surface carries with it the possibility of adsorption, and unsatisfactory penetration to the interior. In such experiments, however, little inhibition of the flavedo enzyme was obtained. Per- haps the solid tissues are so much richer in the regenerating factor than are extracts thereof that the HETP-inhibited enzyme is almost immediately regenerated, and even the DFP-inhibited enzyme recovers slowly. Other- wise the two kinds of recovery must differ fundamentally.

SUMMARY

1. A calorimetric method for the assay of acetylesterase based on the rate of liberation of o-nitrophenol from o-nitrophenyl acetate has been developed. The affinity of acetylesterase for this substrate was found to be much greater than that for acetic esters of aliphatic alcohols.

2. Of the enzymes of plant origin, urease, papain, crystalline p-amylase, pectinesterase, and citrus acetylest,erase, only the acetylesterase was found to be appreciably inhibited by DFP. The pK value for a partially purified citrus acetylesterase was found to be 4.3. For a wheat germ concentrate, the pK value was the same, thus giving additional evidence that the wheat enzyme is acetylesterase and not lipase. Fluoride ion was without effect on acetylesterase; hence the effect of DFP was not due to its hydrolysis to fluoride ion.

The acetylesterase in citrus flavedo press-juice required approximately 10 times as much DFP to cause 50 per cent inhibition as did the enzyme after partial purification from this source.

3. A study of the kinetics of inhibition by DFP showed the reaction to be bimolecular. Hence, to define DFP inhibition with acetylesterase, the time as well as the concentration must be considered.


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4. The inhibition of acetylesterase by DFP was found to be independent of the pH of the reaction mixture over the range of stability of the enzyme (pH 4.9 to 7.4).

5. Eserine was without effect on the hydrolysis of acetylcholine by acetylesterase, and likewise did not protect the enzyme from DFP inhibition.

In the presence of substrate (5 per cent triacetin) 1 X 10e3 M DFP required 40 minutes to reduce the activity to one-half, whereas in the absence of substrate this amount of inhibition occurred in 1 minute. In the presence of substrate at lower DFP concentrations, this same decrease occurred in approximately the same time. Hence the point of attack of the DFP must involve the site of attachment of the enzyme to the substrate.

6. The pK value for inhibition of acetylesterase by HETP (a mixture) was found to be one-fiftieth to one-twenty-fifth of the value for inhibition by DFP. TEP, the active insecticidal constituent of the mixture, was just as effective on a molar basis. EMP, the other major constituent of HETP, was considerably less effective. The reaction of these inhibitors with acetylesterase was likewise bimolecular.

7. Dialysis of acetylesterase inhibited with DFP failed to cause any regeneration of activity. TEP- or HETP-inhibited acetylesterase slowly regenerated on storage at 5”. Sometimes more than 50 per cent of the original activity returned in 30 days. A preparation of citrus pectinesterase accelerated this regeneration.

8. Citrus acetylesterase inhibited in. situ by DFP was found to “regenerate” when the intact fruit was allowed to stand for 3 to 4 days. Hence factors responsible for the regeneration of DFP- and TEP-inhibited acetylesterase exist in citrus fruit.

BIBLIOGRAPHY

1. Adrian, E. D., Feldberg, W., and Kilby, B. A., Rep. Great Britain Ministry SuppE1/, XZ, 3 (November, 1942).

2. Webb, E. C., Biochem. J., 42,96 (1948). 3. Mendel, B., Mundell, D. B., and Rudney, H., Biochem. J., 37,473 (1943). 4. Mazur, A., and Bodansky, O., J. Biol. Chem., 163,261 (1946). 5. Hawkins, R. D., and Mendel, B., Brit. J. Pharmacol., 2, 173 (1947). 6. Jansen, E. F., Nutting, M.-D. F., and Balls, A. K., J. Biol. Chem., 170,417 (1947). 7. Nachmansohn, D., and Feld, E. A., J. Biol. Chem., 171,715 (1947). 8. Nachmansohn, D., Rothenberg, M. A., and Feld, E. A., Arch. Biochem., 14, 197

(1947). 9. Koelle, G. B., J. Phurmacol. and Exp. Therup., 88, 232 (1946).

10. Mazur, A., J. BioZ. Chem., 164, 271 (1946). 11. DuBois, K. P., and Mangun, G. H., Proc. Sot. Exp. Biol. and Med., 64,137 (1947). 12. Jansen, E. F., Jang, R., and MacDonnell, L. R., Arch. Biochem., 15, 415 (1947). 13. Van Slyke, D. D., and Archibald, R. M., J. Biol. Chem., 154,623 (1944). 14. Balls, A. II., Lineweaver, H., and Schwimmer, S., Ind. and Eng. Chem., 32, 1277

(1940).


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A. K. BallsEugene F. Jansen, M.-D. Fellows Nutting and

TETRAETHYL PYROPHOSPHATEFLUOROPHOSPHATE AND

ACETYLESTERASE BY DIISOPROPYL THE REVERSIBLE INHIBITION OF

1948, 175:975-987.J. Biol. Chem.

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of · was not affected by dfp,5 the inhibition of the wheat enzyme would offer additional evidence...

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