CLIN. CHEM. 24/7, 1177-1 181 (1978)

Biochemical Characterization of an Aryl Acetic Ester Hydrolase Isolated from Human Monocytes William K. W. Lam, Julia Chen,’ Edwin Taft,2 and Lung T. Yam3

enzyme is specific for acetyl esters of aromatic alcohols.

nocytic leukemia, total leukocyte count 358 0004zl, was admitted in a semiconscious state. Because of the clinical impression of leukostasis syndrome (8), therapeutic leukapheresis with the continuous-flow Aminco Celltrifuge (American Instrument Company, Silver Springs, Md. 20910) was un-

It is inhibited

dertaken,

A carboxylic-ester hydrolase was isolated from the leukocytes of a patient with myelomonocytic leukemia. Its relative molecular mass as estimated by sucrose density-gradient sedimentation is about 70 000. The purified by fluoride,

but insensitive

to eserine or p-

chloromercuriphenylsulfonate. Hydrolysis of 1-naphthyl acetate was optimal above pH 6.0; of o-nitrophenyl acetate, above 8.0. The common catalytic site for the two types of substrates on the enzyme was confirmed by competitive

inhibition

data.

AddItIonal Keyphrases: leukocytes

enzyme activity

.

leukemia

Many enzymes catalyze the hydrolysis of naphthyl esters. The hydrolysis product, naphthol, forms insoluble colored compounds with azo dyes; therefore, these enzymes are easily detected by histochemical methods. Wachstein and Wolf (1) used

“naphthyl AS acetate” (2-acetoxy-3-naphthoic acid to demonstrate esterase activity among different types of cells in smears of bone marrow and blood. Braunstein (2) observed that only monocytes were stainable with naphthyl acetate as substrate. The chloroacyl esters of naphtholnaphthol-AS or naphthol-ASD-stained mast cells and neutrophilic granulocytes but were inactive for monocytes (3-6). Fischer and Schmalzl (7) showed that the monocyte esterase was fluoride sensitive, while granulocyte esterase was fluoride resistant. The optimal procedures for identification of monocytes and for differential diagnosis of leukemia based on esterase staining were described in our previous reports (5, 6). The substrate and inhibitor specificity of monocyte esterase

aniide)

described above indicate that the high esterase activity of monocytes is due to enzymes that are not present in other leukocytes.The conclusion is strengthened by the electrophoresis of monocyte extract, which shows only one major activityband (6).The activityin thisband was isolatedby ion-exchange chromatography, and its biochemical properties are described in this report.

Materials and Methods Leukapheresis:

A 47-year-old

Departments

of Ophthalmology

man with

acute

myelomo-

and Biochemistry, Albany N. Y. 12208. 2 Department of Hematology, Albany Medical College of Union University, Albany, N. Y. 12208. of Hematology and Oncology, Veterans Administration Hospital, Louisville, Ky. 40207. Received Jan. 27, 1978; accepted May 1, 1978. 1

Medical College of

Union

University,

Albany,

which

reduced

the

white

blood

cell

count

to

38 000/id, by removing about 22 X 1012 cells. Recovery of consciousness followed, and subsequent conventional chemotherapy produced a complete marrow remission. After leukapheresis

the cell suspension

was centrifuged

for

20 mm at 1000 X g, the supernatant fraction discarded, and the packed cells were stored in graduated plastic tubes at -70 #{176}C in 20-mi portions. One tube at a time was thawed as needed to test different purification procedures. This study completed in six months. The chromatographic pattern

was

for

a sample stored at -70 #{176}C for twelve months remained unchanged. Extraction: Twenty milliliters of packed cells was diluted to 100 ml with phosphate buffer (10 mmol/liter, pH 7.5). The suspension was subjected to sonic disruption at 0#{176}C for 3 mm with a Biosonic (Bronwill Scientific Company, Rochester, N.Y. 14603). The probe intensity was set at maximal power. The solubilized proteins were separated from theparticulate fraction by centrifugation (20 000 X g, 15 mm). DEAE-cellulose chromatography: The supernate from the preceding steps was applied to a DEAE-cellulose column (2.5 X 30 cm, Whatman DE-52, equilibrated in phosphate buffer, 10 mmol/liter, pH 7.5). The column was elutedby use of a

linear sodium chloride concentration gradient of 0 to 0.5 mol/liter in a totalof 400 ml ofthephosphate buffer.The eluate was collected in 5-ml fractions, and 10-idportions ofeach fraction were assayed foresteraseactivity, with a-naphthyl acetate or a-naphthyl butyrate as substrate. The sodium concentration was determined in a flame photometer (Coleman Instruments Division, Maywood, Ill. 60153; Model 5lCa). CM-Sepharose chromatography: The active fractions corresponding to the first activity peak oftheabovestepwere combined. Ammonium sulfate was added to 40% saturation (243 mg crystalline ammonium sulfate per milliliter of sample). The precipitated protein, removed by centrifugation, was redissolved in 3 ml of the phosphate buffer. The ammonium sulfate concentration in the supernatant fraction was then brought to 65% saturation (168mg of crystallineammonium sulfate per milliliter of supernatant fraction). The protein precipitated between 40 and 65% saturation withammonium sulfate was dialyzed overnightatO #{176}C against 2 liters of acetate buffer(10 mmol/liter,pH 5.0), then transferred to a CMSepharose column (0.9 X 120 cm, equilibrated with theacetate buffer). The column was eluted by use of a linear concentration gradient of NaCl solution, 0.32 to 0.50 mol/liter, in 400 CLINICAL CHEMISTRY,

Vol. 24, No. 7, 1978

1177

ml of the phosphate buffer. The eluate was collected in 3-ml fractions, and 10-id portions from each fraction were analyzed for esterase activity, with a-naphthyl butyrate or a-naphthyl acetate as substrate. Fractions under the second peak (which was inactive to a-naphthyl butyrate, see Fig. 3, ib) were combined and the enzyme was precipitated by ammonium sulfate (40-65% saturation) and dialyzed. The CM-Sepharose chromatography

step was repeated in the same manner. Electrophoresis was done as described previously (6). For spectrophotometry, we used a final volume of 1 ml, including phosphate buffer (0.1 mol/liter, pH 7.5) and substrate (1 mmollliter). The p-nitrophenol formed was calculated from the absorbance measured at 410 nm. The naphthol formed was estimated from the color produced by adding 2 ml of a color reagent prepared by dissolving 30mg of Fast Garnet GBC, 4.8 g of lauryl sulfate, 0.93 g of sodium acetate, and 2.35 g of sodium barbital in 100 ml water, which is then mixed with 40 ml of 0.2 mol/liter HC1. The color developed with a-naphthol was measured at 570 nm; that developed with fl-naphthol was measured at 520 nm. One unit (U) of enzyme activity is the activity producing hydrolysis of 1 lLmOl of substrate per minute in 1 ml of assay medium. Protein concentration was determined by the method of Lowry et al. (9), with bovine serum albumin as standard. The relative molecular mass of the esterase was estimated by the sucrose density gradient sedimentation method described by Martin and Ames (10).

Results The distribution of esterase activity at different stages of purification is summarized in Table 1. Most of the esterase activity in the cell suspension- was recovered in the soluble fractions after sonic disruption. The activity in the extract was divided into three peaks by DEAE-cellulose chromatography (Figure 1). The first peak was purified by repeated chromatography on a CM-Sepharose column for biochemical characterization. Distribution of activity in the fractions from DEAE-column chromatography was first determined spectrophotometrically. All of the proteins with low affinity for the column emerged from the column in a broad peak (I) as the extract passed through the column. The esterase within peak I reacted to both cx-naphthyl acetate and a-naphthyl butyrate. As sodium chloride concentration was increased above 0.1 mol/liter a sharp activity peak (peak II) was observed. The enzyme activity in this location had a stronger reactivity toward a-naphthyl butyrate than a-naphthyl acetate.

Table 1. Analytical Recovery of Esterase Activity During Purification of the Aryl Acetic Ester Hydrolase Specific activity, U/mg protein Purification stop

Naphthyi acatato

Naphthyl butyrat.

0.044 0.040

0.034

Protsin, mg

1. Sonic disruption

a. soluble fraction b. particulate 2. DEAE-column

0.019

1668 1234

chromatog.

Peak I

0.023 0.039

saturation) 3. 1st CM-column (40-50%

0.017

657

0.024

302

Peak IA

1.832

2.460

2.14

Peak lB

0.919 4.610

0.032

6.79

none

0.35

4. 2nd CM-column

All assays were done In 1 ml at 37 #{176}C, wIth 1 mmol/ilter

substrate.

The third peak had relatively little reactivity toward anaphthyl butyrate as compared to naphthyl acetate. As described earlier (2), a crude extract of leukocytes showed only one electrophoretic band, strongly reactive toward a-naphthyl acetate (Figure 2A), but only weakly toward a-naphthyl butyrate (Figure 2B). The same phenomena were observed in the enzymic activity corresponding to Peak I (Figure 2C, D). The second peak showed a sharp electrophoretic band (Figure 2E) in the same location as that for Peak I and a diffused activity band with slower electrophoretic mobility. The sharp band contained material that reacted with either a-naphthyl acetate or a-naphthyl butyrate (Figure 2F). The third peak showed two bands, which reacted only toward naphthyl acetate (Figure 2G and H). Our present interest is in Peak I only. It was mentioned earlier that spectrophotometric analyses of Peak I showed similar reactivity toward a-naphthyl acetate and a-naphthyl butyrate. However, electrophoretic analysis showed much stronger reactivity toward naphthyl acetate (Figure 2C) than toward naphthyl butyrate (Figure 2D). This was explained later by the results of chromatography on CM-Sepharose. Peak I (Figure 1) was further separated into two peaks (IA and IB) on the first CM-Sepharose column (Figure 3). According to spectrophotometric analyses, Peak IA is slightly

16

12 =

1.0

z

(I)

I,I

z 0

0.5

3.4

‘,

.:f, 50

.1

V( 100

150

FRACTION NUMBER. 4 mI/fraction

#{149} ..

.r

W.

Fig. 1. Results of DEAE-cellulose chromatography The soluble protein fraction fromthesonicdisruption step was transferred to

FIg. 2. Electrophoretic ofpurification

thecolumn,andeluted asdescribed inthetext. The enzyme activity was separated Into three groups,I, II, and Ill. Activity In the eluate was determined spectrophotometrically, with a-naphthyl acetate (-) or a-naphthyl butyrate (- - -) as substrates.Protein concentration is indicated by(

Crudeextract (A, B). DEAE-cellulose chromatography (cf FI9ure 1)fractions 41 (C, 0), 68 (E, F), 89 (G,H),and 50 (K, L). A, C, E, G, land Kwere stainedwith use of a-naphthyl acetate as substrate; B, D, F, Jand L with a-naphthylbutyr-

1178

CLINICAL CHEMISTRY. Vol. 24, No. 7, 1978

ate

patterns of esterases

at different

steps

0.8 -

500

. 0.6

0.4 x =

-

400

300

200 .0.2 100

0

10

20

30

40

50

60

FRACTION NUMBER. 4 mt/fraction

Fig.3.ResultsofCM-column chromatography

4

The active fractions underPeak I (Figure 1) were combIned, precIpitated wIth

ammonium sulfate, and transferredtoa CM-Sepharose column asdescribed in the text. Two active peaks (IA and IB) were observed. IA was active for both acetyl (0) and butyl (#{149}) esters. lB was active only on acetyl ester.

8

10

A ff

#{149} 5

ig. . ec 0 p on es eraseac vi Two different substrates, o.nitrophenyl acetate (0)anda-naphthyl acetate (#{149}), wereassayed atdIfferent pH’s as IndIcated Inthetext.

more reactivetoward a-naphthyl butyrate than a-naphthyl acetate. When material corresponding to Peak IA was subjected to electrophoresis,no activity band was observed (Figure 2,I and J). Peak IA must be a labile enzyme. Electrophoresisof Peak lB showed one strong activity band, reactive only toward a-naphthyl acetate (Figure 2K, L).

1.5

Rechromatography of Peak lB on another CM-Sepharose column yielded a single activity peak with much improved specific activity. The active fractions were combined for biochemical characterization. The purified enzyme is specific for acetyl esters of aromatic alcohols. The acetyl group cannot be replaced by a carboxyl group with more than two carbons. The alcohol group can be either naphthol or nitrophenol. Inactive substrates tested include acetylcholine, naphthyl-ASD-chloroacetate, anaphthyl-leucylamide, o -nitrophenyl propionate, o -nitrophenyl sulfate, o-nitrophenyl-a-D-glucoside, p -nitrophenyl-N-acetyl-fl-D-glucosamide, o-nitrophenyl caproate, and a-naphthyl palmitate. The Km and Vm of the active substrates are shown in Table 2. The pH-activity relationship is shown in Figure 4. When o-nitrophenyl acetate is used as substrate the enzyme has very little activity below pH 6 and maximal activity above pH 8.0. The optimal pH for the hydrolysis of a-naphthyl acetate is shifted to the acidic side. p -Nitrophenyl acetate is very unstable above pH 7.5, therefore Figure 5 shows only the results for o-nitrophenyl acetate. The different pH optima of the two substrates led us to wonder if the enzyme preparation contained two protein species, one acting on each of the two different substrates. This possibility was ruled out by the competitive inhibitory effect of o-nitrophenyl acetate (Figure 6) on the hydrolysis of a-naphthyl acetate. When o-nitrophenyl acetate was added to the assay medium for a-naphthyl acetate, naphthol pro-

6

p11

__.

1.0

0.5

0.2

0.4

0.6

0.8

1/S (mol/liter)

Fig. 5. Competitive inhibition by o-nitrophenyl acetate (2 mmol/liter) ofhydrolysis ofnaphthylacetate V = mol of substratehydrolyzedper minute,as measured by the colorimetric methodfor naphthol. Control samples (#{149}) containedonly naphthyl acetate. The upper curve shows the inhibitory effect ofo-nitrophenyl acetate, 2 mmol/llter, (0)on thehydrolysis ofdifferent concentrations ofnaphthyl acetate

1/v

0.

Table 2. KinetIc Properties of Aryl Acetic Ester Carboxylic Ester Hydrolase Substrate p-Nitrophenyl acetate

o-Nitrophenyl acetate a-Naphthyl acetate 3-Naphthyl acetate

Km

1.70± 1.30 0.95± 0.10 1.34 ± 0.14 1.84± 0.51

Vmax

2.68 ± 1.1 11.8± 0.5 14.5± 1.0 8.6 ± 1.5

Allassays were done In 1 ml at 37 #{176}C. At least 10different substrate concentrations(0.1 to2.0mmol/Ilter) were used. The mean value andstanderd error was calculated by use of the computer program described by Bliss andJames (22).

-1.0

1.0

2.0

0-Ni trophenyl acetate ,nfl

Fig.6. Estimation of inhibition constantof o-nitrophenyl ace-

tate Two concentrations of a-naphlhyl acetate, 0.3 mmol/llter (0) and1.0 mmol/Ilter (#{149}), were assayedInthepresence ofdIfferent concentrationsof o-nitrophenyl acetate

CLINICAL CHEMISTRY, Vol. 24, No. 7, 1978

1179

-20

20 40 FLuORIDE. ,1/Iter

60

FIg.7. Estimationof inhibition constantoffluoride Two concentrations ofa-naphthyl acetate, 0.3 rmRol/ilter (0).and1.0mrnoi/lrter (C).wereassayed inthe presence of different concentrations of fluoride

duction was competitively inhibited by o-nitrophenyl acetate, which indicated that both substrates compete for the same active site of one enzyme. The K for nitrophenyl acetate, estimated by the graphical method of Dixon (11), was about 1 mmol/liter, which is very similar to the Km value when it was used as the substrate (Figure 6). The enzyme was insensitive to 0.1 mmol/liter p-chloromercuriphenylsulfonate and eserine. Competitive inhibition by fluoride was observed (K1 = 12 mmol/liter) (Figure 7). Centrifugation of a mixture of the purified enzyme and hemoglobin showed similar sedimentation properties for the two proteins. The esterase activity sedimented slightly faster than that of hemoglobin (Figure 8). We estimate the relative molecular mass of the esterase to be about 70 000.

Discussion Carboxylic-ester hydrolase includes many enzymes that share similarcatalytic activity toward synthetic substrates. These enzymes have been reviewed by Latner (12), Krisch (13), Shnitka (14), and Masters and Holmes (15). Because there are so many of these enzymes and because of the overlapping substrate specificity, their subdivision has been difficult.

90.8

cO.6 C, a

O.4

#{149} 3o C

#{149} 20 C,

8 0 50.2

#{149} ,1O =

=

10

15

FRACTION NUMBER

FIg. 8. Relative molecular

mass estimation by sucrose density

gradient sedImentation Thepurified hydrolase (80 U in0.2ml)was mixedwith hemoglobin and layered onto a sucrose solution (sucrose concentrationvaried from 50 to250 g/lfter; the total volume of sucrose was 5 ml)andcentrifuged for 23 hat 40000rpm ina Beckmanswinging-bucket rotor(BeckmanInstruments, Inc., Fullerton, Calif. 92634). The sucrose solutionwas divided Into 23 fractions by puncturing the bottom of the tubes. Aliquots of 10 zI of each fractionweremeasured for esterase actIvIty (5). For hemoglobin determinatIon (0), 50-il allquots, diluted 10-fold, were measured at 410 nm

1180 CLINICALCHEMISTRY.Vol. 24, No. 7, 1978

The requirement of an aromatic alcohol group for the present enzyme favors its classification with the group of aryl-ester hydrolases. This group of esterases has been isolated from serum (16, 17) and liver(18-20), but it was not specific to acetyl esters. On the other hand, the hydrolases specific to aceticesters, described by Bergman et al. (21) and Jasen et al. (22), were not specific tothearomaticalcoholgroup.The present report describes an enzyme specificity requiring both the acetyland aryl groups. Therefore, it should be classified as an aryl acetic ester hydrolase (EC 3.1.1.2). The lack of reactivity toward carboxylic esters of choline and the insensitivity to eserine clearly rule outany relationship ofthis enzyme to choline esterases. The unique substrate and inhibitor specificity, as well as its electrophoretic mobility, clearly show that the enzyme described in this report is a distinct protein species, different from thosedescribedpreviously(12-22). The high esterase activity observed in one type ofcells could be the resultof a highconcentration ofallesterases, or to a disproportionate activity of one or more specific enzymes related tothe specialized function ofthecell. Previous studies show that monocyte esterase has properties different from those of other leukocytes. The biochemical characteristics of thepurifiedenzyme also point up the unique features of monocyte esterase. Therefore, the high esterase activity in monocytes clearly is attributable to the occurrence of a few specific enzymes rather than to a general increasein all esterases, as the name “nonspecific

esterase”

implies.

The criteria for “nonspecific esterase” previously observed in monocytes were sensitivity to fluoride and insensitivity to eserine inhibition (6, 7). Enzymes with these properties were observed by electrophoresis among the cationic proteins. On staining for enzyme activity with a-naphthyl-ASD-chloroacetate, at least nine bands could be seen in the extractof normal leukocytes, but only band 5 was reactivetoward naphthyl acetate (6). It is the major band shown by monocytes, but is absent from granulocytes and lymphocytes. Our previous report (6) showed that when the acidity of the staining solution was reduced to pH 6.5, the intensity of band 5 was unchanged, while the other bands disappeared. This was consistent with the pH optimum-curve of the purified enzyme described

in Figure

4, which shows the same activityfor

naphthyl acetatebetween pH 6 and 8.Allpropertiesofthe purified enzyme described in this report, including electrophoretic mobility and substrate and inhibitor specificity, are identical withthe previousdatafor“nonspecific esterase” of monocytes. This study has been reproduced infour cases of myelomonocytic leukemia, and one case of acute monocytic leukemia. The enzyme was barelydetectableinthreecasesofchronic granulocytic leukemia, and not detectable in two cases of chronic lymphocytic leukemia. The traceamount of band 5 observed in leukocytes of granulocytic leukemia is probably due tocontaminationby monocytes in the preparation. The number of monocytes in lymphocytic leukemia is usually negligible. Our column chromatographic studies demonstrate the presence of two esterases (Peaks I and II) with identical electrophoretic mobility. Both are reactive toward a-naphthyl acetate, but only the activity corresponding to Peak II isreactive toward a-naphthyl butyrate. Another esterase, Peak IA (Figure 3) is inactive after electrophoresis (Figure 21, J). Therefore, electrophoresis of the crude extract shows only one band, with strong reactivity toward naphthyl acetate but weak reactivity toward naphthyl butyrate. The characterization of Peak IA and II will be described in the future. This study was supported in part by NIH Grants EY 01464 and by the Medical Service of the Veterans Administration Fund No. 6039850.

References 1.

Wachstein,

M., and Wolf,

D., The histochemical

demonstration

of esterase activity in human blood and bone marrow smear.J. Histochem. Cytochem. 6,457 (1958). 2. Braunstein, H., Esterase in leukocytes. J. Histochem. Cytochem. 7,202(1959). 3. Gomori, G., Chioroacyl esters as histochemical substrates. J. Histochem. Cytochem. 1, 469 (1963). 4. Moloney, W. G., McPherson, K., and Fliegelman, L., Esterase activityin leukocytesdemonstrated by the use of naphthol AS-D chloroacetate substrate. J. Histochem. Cytochem. 8,200 (1960). 5. Yam, L. T., Li, C. Y., and Crosby, W. H., Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283 (1971). 6. Li, C. Y., Lam, K. W., and Yam, L. T., Esterases in human leukocytes. J. Histochem. Cytochem. 21, 1(1973). 7. Fischer, R.,and Schmalzl, F., Uber die Hemmbarkeit der Esterase Aktivitat in Blutmonocyten durch Natrium Fluorid. Kim. Wocherzschr. 42,751 (1964). 8. McKee, L. C., and Collins, R. D., Intravascular leukocyte thrombi and aggregates as a cause of mobidity and mortality in leukemia. Medicine 53,463 (1974). 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265 (1951). 10. Martin, R. G., and Ames, B. N., A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. Biol. Chem. 236, 1372 (1961).

11. Dixon, M., The determination of enzyme inhibitor constants. Biochem. J. 55, 170 (1953). 12. Latner, A. L., and Skillen, A. W., Isoenzymes in Biology and Medicine. Academic Press,New York, N.Y., 1968,p 66. 13. Krisch, K., Carboxylic ester hydrolases. In The Enzymes, 5, P. D. Boyer, Ed., Academic Press, New York, N. Y., 1971, p 43. 14. Shnitka, T. K., Esterases-Non-specific esterases. In Electron Microscopy of Enzymes, Principles and Methods, 3, M. A. Hayat, Ed., Van Nostrand Reinhold Co., New York, N. Y., 1974,p 1. 15. Masters, C. J., and Holmes, R. S., IV, Esterases, isoenzymes, multiple enzymes forms and phylogeny.In Advances in Comparative Physiology and Biochemistry, 5,0. Lowenstein,Ed., Academic Press, New York, N. Y., 1974,p 141. 16. Main, A. R., The differentiation of the A-type esterases insheep serum. Biochem. J. 75, 188 (1961). 17. Aidridge, W. N., Some esterases of the rat. Biochem. J. 57, 692 (1954). 18. Ecobichon, D. J., Carboxylesterase isoenzymes in rabbit liver. Isoenzyme 1, 389 (1975). 19. Arndt, R., and Krisch, K., Catalytic properties of an unspecific carboxylesterase from rat-liver microsomes. Eur. J. Biochem. 36, 129 (1973). 20. Hayase, K., and Tappel, A. L., Microsomal esterase of rat liver. J. Biol. Chem. 244, 2269 (1969). 21. Bergman, F., Sega!, R., and Robinson, S., Fractionation of Cesterasefrom the hog’s kidney extract. Biochem. J. 77, 209 (1960). 22. Jasen,E. F., Jang, R., and Mackonnell, L. R., Citrus acetylesteraBe. Arch. Biochem. 15,415 (1947).

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CHEMISTRY, Vol. 24, No. 7, 1978

1181