[45] Carboxylesterases-amidases

[451

333

CARBOXYLESTERASES-AMIDASES

Acknowledgment Substantial parts of the author’s work were supported by the Deutsche Forschungsgemeinschaft (Grant We 68615). A. Grossmann and W. Lijdige contributed many details to the enzyme preparation.

[45] CaFboxylesterases-Amidases By EBERHARD

HEYMANN

and ROLF MENTLEIN

Rat liver contains a number of carboxylesterases (EC 3.1.1.1) of the serine hydrolase type. I-3 Their physiological role remains unknown, although most are capable of cleaving monoglycerides of long chain fatty acids’ and, therefore, can also be classified as monoacylglycerol lipases (EC 3.1.1.23). Because most rat liver carboxylesterases are also active on aromatic amides,’ we use here the term “carboxylesterase/amidase” and list the isoelectric point (e.g., pZ 6.0) to discriminate between isoenzymes or multiple forms. This preliminary nomenclature seems to be more convenient than others that have been used before. Table I both summarizes the literature on purification procedures and compares the nomenclature used by the authors. Here we report the simultaneous purification of five of the most prominent rat liver carboxylesterases-amidases. All of these enzymes are found in the microsomal fraction. As a side product of the procedure described, a dipeptidyl aminopeptidase (EC 3.4.14.--; dipeptidyl aminopeptidase IV, postproline dipeptidyl peptidase) is obtained that also belongs to the group of serine hydrolases.’ Assay

Methods

For an optimal discrimination between the various carboxylesterases, the three substrates (methyl butyrate, 4-nitrophenyl acetate, and acetanilide) should be used throughout the isolation procedure although it is not necessary to test every fraction with each substrate. The acetanilide assay is described in Article [521 of this volume. ’ R. Mentlein, S. Heiland, and E. Heymann, Arch. Biochem. Biophys. 200, 547 (1980). ’ E. Heymann, in “Enzymatic Basis of Detoxication” (W. B. Jakoby, ed.), Vol. 2, p. 291. Academic Press, New York, 1980. 3 W. Junge and K. Krisch, CRC Crir. Rev. Toxicd. 3, 371 (1975). 4 E. Heymann and R. Mentlein, FEBS Left. 91, 360 (1978).

METHODS

IN ENZYMOLOGY,

VOL. 77

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O-12-181977-9

334

ENZYME

PREPARATIONS

TABLE CORRELATIONOFTHENOMENCLATURE AND REPORTS

[451

I

FORRATLIVERCARBOXYLESTERASES-AMIDASES or4 PURIFICATION PROCEDURES

Enzymes in the nomenclature of this report Other reports

Carboxylesterase, pz 5.2’s”

Earlier reports from our laboratory”**” Ljungquist and AugustinssotP Akao and Omurae Haugen and Suttie’ Ikeda et al. g*h Raftell et al. i Kaneko et al.’ Ishitani et al. k Oerlemans et al. ’

Carboxylesterase, pz 5.t.P” Esterasel amidase EAJEA,”

Carboxylesterase, pZ 6.0”

Carboxylesterase, pZ 6.2”

Esterase El0

Esterase EzP

Esterase al(=eljm

Carboxylesterase, pZ 6.4”

Esterase b,” e3

e3

Amidase”

Esterase I0

Esterase Aa

Esterase B,’

Esterase II”

Monoacylglycerol lipase’

Esterase e,* Esterase LI’ Esterase E- 1u Glycerol monoester hydrolase’

a R. Arndt and K. Krisch, Hoppe-Seyler’s Z. Physiol. Chem. 353, 589 (1972). * R. Arndt, E. Heymann, W. Junge, and K. Krisch, Eur. J. Biochem. 36, 120 (1973). c R. Arndt, H.-E. Schlaak, D. Uschtrin, D. Siidi, K. Michelssen, and W. Junge, HoppeSeyler’s Z. Physiol. Chem. 359, 641 (1978). d A. Ljungquist and K.-B. Augustinsson, Eur. J. Biochem. 23, 303 (1971). e T. Akao and T. Omura, J. Biochem. (Tokyo) 72, 1245 (1972). ‘D. Haugen and J. Suttie, J. Biol. Chem. 249, 2717 (1974). g Y. Ikeda, K. Okamura, T. Arima, and S. Fujii, Biochim. Biophys. Acta 487, 189 (1977). h Y. Ikeda, K. Okamura, and S. Fujii, Biochim. Biophys. Acta 488, 128 (1977). i M. Raftell, K. Berzins, and F. Blomberg, Arch. Biochem. Biophys. 181, 534 (1977). ‘A. Kaneko, Y. Yoshida, K. Enomoto, T. Kaku, K. Hirate, and T. Onoe, Biochim. Biophys. Acta 582, 185 (1979). h R. Ishitani, H. Kin, T. Kuwae, K. Moroi, and T. Satoh, Jpn. J. Pharmacol. 29, 413 (1979). f M. C. Oerlemans, M. M. Geelhoed-Mieras, and W.‘C. Hiilsmann, Biochem. Biophys. Res. Commun. 78, 1130 (1977). m--I’Substrates used for assay during purification: m Cnitrophenyl acetate: ” acetanilide; D methyl butyrate; P 2-nitrophenyl acetate; q phenyl butyrate; r I-monooleylglycerol; s I-naphthyl propionate; ’ 2-naphthyl acetate; ” isocarboxazide.

1451

CARBOXYLESTERASES-AMIDASES

335

Hydrolysis of Methyl Butyrate

In weakly buffered solutions, the amount of butyric acid released by enzymatic hydrolysis can be recorded continuously by the pH-stat technique.j Five milliliters of 5 mM methyl butyrate6 are pipetted into a stoppered vessel thermostatted to 30”. The pH is adjusted to 8.0 and maintained by continuous addition of 0.04 M NaOH from a 0.25-ml burette operated by an automatic titrator (e.g., TTT 1 from Radiometer, Copenhagen, Denmark, with autoburette ABU 13 and recorder). A blank, caused by spontaneous hydrolysis and COz adsorption, is recorded for 3 min. Then the enzymatic reaction is started by addition of lo-500 ~1 of sample. One unit of carboxylesterase activity corresponds to 1 pmol of NaOH consumed per minute. The NaOH consumption is linear with time up to at least 10 pmol. With methyl butyrate concentrations above 2 mM, carboxylesterase pZ 6.0 shows substrate inhibitions However, the activities obtained with the 5 mM standard substrate are less than 10% below those obtained with the optimal 2 mM solution. Hydrolysis of 4-Nitrophenyl

Acetate

The yellow phenol liberated from 4-nitrophenyl acetate at alkaline pH is observed spectrophotometrically at 405 nm’ in a filter photometer (e.g., Eppendorf, Hamburg, FRG) or spectrophotometer (e.g., Hitachi 100-40, Tokyo, Japan). This assay is well suited for on-line registration and calculation with a computer (e.g., Commodore 3032, Palo Alto, CA) equipped with a digital voltmeter (e.g., Micrologic 415, Miinchen, FRG). Another advantage of the assay is that all rat liver carboxylesterases cleave this ester. ’ A 0.5 mM solution of 4-nitrophenyl acetate is made daily by dissolving 18.1 mg of the ester in 1 ml acetonitrile and adding water to 100 ml. Portions of 1.8 ml of the substrate solution at 30” are pipetted into a thermostatted cuvette with an optical path length of 1 cm. After addition of 0.2 ml of 0.5 M Tris-HCl, pH 8.0, the blank caused by spontaneous hydrolysis is registered for 60 sec. The enzymatic reaction is started by addition of lo-100 ~1 of sample; the release of 4nitrophenol is registered for an additional 30 to 60 sec. If a recorder is used instead of the computer, the period of recording should be twice the time period mentioned. The increase in absorbance is linear with time up to a difference of 1.O. For the calculation, a molar absorbance of 16,400 liters mol-’ cm-’ is used. ’ R. Am&, E. Heymann, W. Junge, and K. Krisch, Eur. J. Biochem. 36, 120 (1973). ’ The ester is dissolved in water because traces of most organic solvents greatly influence the catalytic activity. ’ Adapted from K. Krisch, Biochim. Biophys. Acta 122, 265 (1966).

50

FIG.

- Sephacel hydrolyzing

4

Sephodex

fraction

Ammomum

G - 150

sulfate

Step

5b

I

DEAE-

Sephacel

Tusjng

preclpitohon

of five rat liver carboxylesterases

l~oc”,;“g

step

sulfate

1. Flow chart of procedure for simultaneous purification

Acetonllideactivity

DEAE

Methylbutyrate-hydrolyzmg

Step “I-.-.

step

3

step

Extractmn

suspension

Solubillsate

Ammonium

2

step

Microsomal

WI

337

CARBOXYLESTERASES-AMIDASES

Protein Estimation In general, the absorbance at 280 nm provides a sufficient basis for the estimation of protein concentration during the purification procedure. We use a factor of 0.72 that has been obtained with a highly purified pig liver carboxylesterase.8 AzsOmeasured with a l-cm light path and multiplied by the factor, gives the protein concentration in mg/ml. For the estimation of protein in microsomes, we use the biuret procedure as described by Alt et a1.g Procedure for the Simultaneous Carboxylesterases’

Purification

of Five Rat Liver

General. Figure 1 presents a general survey of the procedure. All steps are performed at temperatures of 0” to 4”. The columns for chromatography or isoelectric focusing are thermostatted at 4”. Although the procedure was developed with livers of male Wistar rats, similar results are obtained with females of this strain, and from male or female SpragueDawley rats. All gels used for preparative chromatography are prepared, equilibrated, and regenerated as described by the manufacturer (Pharmacia) . Step I. Isolation of Microsomes. The procedure for the isolation of microsomes is essentially that of Krisch.‘O Male Wistar rats of 150-250 g, fasted for 24 hr, are killed by decapitation. The livers of 50-100 rats are collected in ice-cold 0.25 M sucrose. Fat and connective tissues are removed. To 150 g of liver, 500 ml of 0.25 M sucrose are added and the mixture is homogenized with a blender (e.g., Ultra-Turrax T 45/6, Janke und Kunkel, Staufen, FRG) at a rotatory speed of 10,000 rpm for 1 min, (the distance of the prongs that rotate in a shell of knives is 37 mm). The homogenate is diluted to 1 liter with 0.25 M sucrose, homogenized for another 20 set, and centrifuged for 30 min at 16,000 g . The sediments are discarded and the supernatant liquid is centrifuged for 60 min at 105,000 g . Using a glass homogenizer with Teflon pestle, the sediment (microsomes) is suspended in 0.1 M Tris-HCl at pH 8.5 so that 1 g of suspension corresponds to 2 g of fresh liver. This microsomal suspension is kept at -20 until the enzyme isolation starts with Step 2. Step 2. Extraction. The microsomal suspension, 125 ml, is thawed and treated in an ice bath with the blender described in Step 1 for 5 x 30 set in order to reduce the size of the microsomal particles.” Pauses of 90 set are 8 M. Kunert and E. Heymann, FEES Lerf. 49, 292 (1975). ’ J. Alt, K. Krisch, and P. Hirsch, J. Gen. Microbial. 87, 260 (1975). lo K. Krisch, Biochem. Z. 337, 531 (1963). ” E. Heymann, W. Junge, K. Krisch, and G. Marcussen-W&T, Hoppe-Seyler’s Gem. 355, 155 (1974).

Z. Physiol.

338

ENZYME

TABLE GRADIENTS

[451

PREPARATIONS

II

FOR STEPS 5~ AND

Chamber no.

1 M NaCl in 10 mM TrisHCl, pH 8.0 (ml)

10 mM Tris-HCl, pH 8.0 (ml)

1 2 3 4

12 28 60 160

388 372 340 240

5~

Resulting NaCl concentration (mW 30 70 150 400

necessary between each 30-set pulse to avoid increasing the temperature above 10”. To the mixture are added 250 ml of 0.1 M Tris-HCl at pH 8.5, containing 3.75 g of saponin (Merck, Darmstadt, FRG), and the suspension is stirred for 60 min (final concentration: 1 g saponin/lOO ml). After centrifugation of 2 hr at lO$OOOg, the supematant fluid contains the bulk of the carboxylesterase-amidase activities. The yield of solubilized carboxylesterases-amidases can be improved if digitonin is used instead of saponin. Step 3. Ammonium Sulfate. Within 1 hr of solubilization, 200 ml of ammonium sulfate solution, saturated at 0”, are added dropwise with stirring into 300 ml of the preparation from Step 2. The precipitate is collected by centrifugation (30 min at 10,000 g) and discarded. Solid ammonium sulfate, 103 g, is added in small portions to the supematant fluid. The salt precipitation step should be completed within 60 to 90 min. The final ammonium sulfate concentration (70% saturation) is controlled by titration with BaCl,.‘* After centrifugation (30 min at 15,000 g), the carboxylesterase-containing sediment is dissolved in 15 ml of 10 mM Tris-HCl at pH 8. Step 4. Sephadex G-150. The solution from Step 3 is applied to a gel filtration column (5.0 x 96 cm) filled with Sephadex G-150. The column is equilibrated and eluted with 10 mM Tris-HCl at pH 8. The eluate is collected in fractions of 6 ml that are assayed with both methyl butyrate and 4nitrophenyl acetate. 4-Nitrophenyl acetate activity is found in two peaks in the range of the fractions 60 through 120. The first peak with proteins of higher molecular weight (about fractions 60 to 90) contains the bulk of methyl butyrate-hydrolyzing activity and is further purified in Step 5a. The low-molecular-weight portion (fractions 91 to 115) is retained for Step 5b. Step 5a. DEAE-Sephacel (High-Molecular- Weight Fraction). A column (2.6 x 33 cm) of DEAE-Sephacel is equilibrated with 10 mM Tris-HCl at I* H. V. Bergmeyer, G. 333, 471 (1961).

Holz,

E. M. Kander, H. Miillering,

and 0. Wieland, Biochem. Z.

1451

CARBOXYLESTERASES-AMIDASES

339

pH 8.0 and charged with the high-molecular-weight fraction obtained in Step 4. Four chambers of the gradient former as described by Peterson and Sober13 are filled according to Table II, and the column is eluted with the resulting concave gradient of NaCl. If the volume of the applied enzyme solution is about 100 ml, and if fractions of 9.5 ml are collected, the 4-nitrophenyl acetate-hydrolyzing activity is found at about fractions 58 to 115, methyl butyrate-hydrolyzing activity in fractions 70 to 115, and acetanilide-cleaving enzymes in fractions 90 to 115. Based on the elution-activity profile, three ranges with enzyme activity are pooled. 1. The first 4nitrophenyl acetate-hydrolyzing peak (about fraction 63) contains low amounts of carboxylesterases pZ 6.2 and 6.4. Because the bulk of these esterases is obtained in Step 5b, this preparation may be discarded. 2. The steep methyl butyrate-hydrolyzing peak (about fraction 80) mainly consists of carboxylesterase-amidase pZ 6.0. It is divided from the overlapping acetanilide-cleaving peak at the intersection of the steep decline of the first and the steep incline of the second activity. The pooled fractions are dialyzed against 5 mM Tris-HCl at pH 8.0 (2 x 5 liters) for 18 hr and are kept frozen (-20”) until the further separation in Step 6b. 3. The remaining acetanilide-hydrolyzing peak at about fraction 105 is combined with the corresponding peak of Step 5b. It contains carboxylesterase-amidase pZ 5.6. The descending part of this peak also contains the main portion of the dipeptidyl aminopeptidase IV. Step 5b. DEAE-Sephacel (Low-Molecular-Weight Fraction). The procedure in this step parallels exactly that of Step 5a, except that the lowmolecular-weight portion obtained in Step 4 is applied to the ion-exchange column. When the eluate fractions are assayed with 4-nitrophenyl acetate, activity is found in fractions 55-130 and, with acetanilide, in fractions 80-130. The steep esterase peak at about fraction 63 is pooled, dialyzed as described in Step 5a, and further purified by isoelectric focusing (Step 6a). The acetanilide-hydrolyzing peak at about fraction 105 is combined with the corresponding peak of Step 5a and also dialyzed as previously described. After dialysis, the latter enzyme solution is frozen at -20” until further purified in Step 6c. Step 6. Isoelectric Focusing GENERAL PROCEDURE. An apparatus for carrier-free isoelectric focusing in sucrose gradients of a volume of 440 ml (LKB 8122) is used in Steps 6a-c. The apparatus is filled and emptied with a peristaltic pump at a speed of 50 ml/hr. Only specially purified sucrose “for density gradients” (Merck) is used. The lower electrode (anode) buffer is a mixture of 60 g sucrose, 60 ml water, and 4 ml of 1 M H3P04. Over this, the enzyme and ampholyte-containing sucrose gradient is layered, and the cathode fluid I3 E. A. Peterson

and H. A. Sober,

Anal.

Chem.

31, 857 (1959).

340

ENZYME

PREPARATIONS

[451

(50 mM NaOH) is pumped on top. The gradient is formed with a twochamber gradient mixer (LKB): The first chamber contains a solution made of 108 g sucrose, 5 ml of ampholyte (Ampholine, LKB), and 136 ml water or dialyzed enzyme solution. The second chamber contains a solution of 10.8 g sucrose, 5 ml Ampholine, and 204 ml of the dialyzed enzyme fraction of Step 5. If the volume of the enzyme fraction is less than 204 ml, it is filled up with water; if greater, the excess volume is filled up to 136 ml with water and is applied to the first chamber. The enzymes are focused for l-2 days at increasing voltage (max. 800 V), so that the power applied never exceeds 4 W. Most of the time, the power remains below 2 W. Focusing is terminated if precipitation of one of the enzyme bands becomes visible. The eluate is collected in fractions of 40 drops (about 2.2 ml), and the pH of each is determined at 4”. Step 6a. Isoelectric Focusing of the Carboxylesterases with pi Values > 6. After dialysis, the first carboxylesterase peak of the DEAE-Sephacal column described in Step 5b is applied to the isoelectric focusing gradient. Each chamber of the gradient former contains 4.5 ml of Ampholine pH 5-7 and 0.5 ml of Ampholine pH 3.5-10. The eluate fractions are assayed with 4-nitrophenyl acetate. The largest activity peak is that of the pZ 6.2 enzyme. The fractions in this peak are pooled as are those of the pZ 6.4 enzyme; both are retained for the final gel filtration (Steps 7a and b). The small peak at pZ 6.0 is discarded; it contains carboxylesterase-amidase pZ 6.0 of lesser purity than that obtained in Step 6b. Step 66. Isoelectric Focusing of the Methyl Butyrate Cleaving Enzyme. The dialyzed methyl butyrate-hydrolyzing peak from Step 5a is applied in the same manner and with the same amounts and types of Ampholine as in Step 6a. A single methyl butyrate-hydrolyzing peak is found in the eluate at pH 6.0. This represents carboxylesterase pZ 6.0 and is subjected to gel filtration in Step 7c. Step 6c. Isoelectric Focusing of the Acetanilide-Cleaving Enzymes. The combined acetanilide-hydrolyzing fractions of Steps 5a and b are applied to the isoelectric focusing column as described earlier. Sometimes the total volume of these fractions exceeds the limited sample volume (340 ml) of the apparatus. In that event, the enzyme solutions must be concentrated by lyophilization or ultrafiltration. The gradient contains 10 ml of ampholyte pH 4-6.5, (e.g. Pharmalyte, Pharmacia; or a mixture of 5 ml Ampholine pH 4-6 and 5 ml Ampholine pH 5-7). The eluate fractions of this step are assayed with acetanilide and, if desired, with glycylprolyl-Z naphthylamide14; with the latter substrate a dipeptidyl aminopeptidase peak with a pZ at about 4.8 is found. The acetanilide assay discloses a I4 J. K. McDonald, P. X. Callahan, S. Ellis, and R. E. Smith, in “Tissue Proteinases” (A. J. Barett and T. J. Dingle, eds.), p. 69. Elsevier/ North-Holland Biomedical Press, Amsterdam, 1971.

Isolectric focusing followed by Sephacryl S-200

DEAE-Sephacel

Microsomes Solubilized fraction Salt fractionation Sephadex G-150

Fraction

Low-molecularweight fraction High-molecularweight fraction Esterases pl 5.2 and 5.6 Esterase pI 6.0 Esterases pl 6.2 and 6.4 Esterase pl 5.2 Esterase pl 5’.6 Esterase pl 6.0 Esterase pl 6.2 Esterase pl 6.4

Isoenzyme

-

25 82 6,000 -

11,31@ -

58 30 1.3 3.2 16 5.9 2.6

140

20.950

295 8

33,250 23,750 21,950 800

Methyl butyrate

8750 595 500 165

Protein (md

ACTIVITIES

-

0.71 19.6 -

20.6

5.9

29.5 52.2 47.1 35.3

Acetanilide

18.8 25.6 375.0 5.1 2.0

72 710 550 335

195.0 3.0

17.0

71.0

3.8 39.9 43.9 4.8

Methyl butyrate

1,850 1.700

92

3.700

21,150 11,150 8,200 2.700

4-Nitrophenyl acetate

Total activity (pmol min-I)

TABLE III PURIFICATION OF THE CARBOXYLESTERASE-AMIDASE

-

5.5 22.4 44.4 94.0 131.0

31.9 55.7

0.55 6.13 -

11.5

12.5

2.42 18.8 19.4 16.4

CNitrophenyl acetate

2.58

0.020

0.0034 0.088 0.108 0.255

Acetanilide

Specific activity (Fmol min-’ mg-‘1

342

ENZYME

[451

PREPARATIONS

major peak with pZ 5.6 and a minor one with pZ 5.2. For unknown reasons, the latter peak is sometimes absent. This focusing column always contains some carboxylesterase pZ 6.0 that may be found if the eluate is assayed with methyl butyrate. Step 7u-fl Sephacryl S-200. A column (2.6 x 95 cm) is packed with Sephacryl S-200 (Pharmacia) and equilibrated and operated with 10 mM Tris-HCl at pH 8.0. Each of the six enzymes obtained in Steps 6a-c are subjected to passage through this column. The column is designed to remove ampholyte and sucrose, but also separates remaining traces of the high-molecular-weight enzymes (carboxylesterase pZ 6.0 and dipeptidyl peptidase) from those with lower molecular weight, and vice versa (compare Table V). The yields and specific activities estimated after the individual purification steps are summarized in Table III. Properties of the Carboxylesterases-Amidases Purity of the Enzymes. The five isolated carboxylesterases-amidases have been completely separated from each other as can be demonstrated by analytical polyacrylamide gel electrophoresis and isoelectric focusing on flat gels that are stained for esterase activity (1). It is also evident from these gels that carboxylesterase-amidase pZ 5.6 remains heterogenous; this is supported by an analysis of the terminal amino acids.’ Only the enzymes with pZ 5.6 and pZ 6.0 appear as homogeneous proteins on TABLE SUBSTRATE

SPECIFICITY

OF HIGHLY

IV

PURIFIED

RAT

LIVER

CARBOXYLESTERASES’

Specific activities (Fmol mm’

Acetanilide Butanilicaine” Methyl butyrate 4-Nitrophenyl acetate I-Monobutyryl glycerol l-Monolauryl glycerol I-Monooleyl glycerol

mg-‘)

Esterase pl 5.2

Esterase pl 5.6

Esterase pI 6.0

Esterase pI 6.2

Esterase pl 6.4

0.55 0.0 18.2

6.1 2.0 25.6

0.0 8.3 4100

0.0 0.0 5.1

0.0 0.0 2.0

5.5

22.4

44.4

0.0

8.2

11.9

6.0

0.0

0.0

2.0

8.0

20.5

15.4

0.29

1.59

0.67

(I N-(Butylaminoacetyl)-2-cbloro-dmethylanilide. b At 2 mM substrate concentration.

94

3.21

131

3.55

TABLE PHYSICAL

Esterase pl 5.2 Esterase pl 5.6

Esterase pl 6.0 Esterase pl 6.2 Esterase pl 6.4

343

CARBOXYLESTERASES-AMIDASES

[451

PROPERTIES

OF THE

FIVE

V CARBOXYLESTERASES-AMMDASES’

Relative mobilities in polyaci-ylamide gel electrophoresis (gel concentration = 7.5%)

Subunit weight”

Molecular weight*

0.62

58,000

60,000

Glycine

0.45 0.43 0.41

61,000

60,000’

0.21

58,000

180,000

Glycine, tyrosine, phenylalanine, and others Tyrosine

0.35

61,000

60,000

0.32

61,000

60,008

N-terminal amino acid

C-terminal amino acid

Leucine (and others?)

-Ala-Val-Leu

’ Estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Estimated by gel chromatography. ’ This enzyme associates to trimers at higher concentrations; R. Amdt, H. E. Schlaak, D. Uschtrin, D. Siidi, K. Michelssen, and W. Junge, Hoppe-Seyler’s Z. Physiol. Chem. 359, 641 (1978).

sodium dodecyl sulfate gels. Each of the other carboxylesterase-amidase preparations show minor impurities with this technique. Active site titration with diethyl(4nitrophenyl) phosphate” reveals a purity* of >90% for carboxylesterase pZ 6.0, and of about 80% for the pZ 5.6 enzyme on the basis of a subunit weight of 60,000 and biuret protein estimation.s Stability. A solution of purified carboxylesterase pZ 6.0 shows no significant loss of activity in 6 hr at 30” (pH 8.0). Solutions of all carboxylesterases-amidases may be kept at - 18” without loss of activity for months. Inhibitors. Active site-directed inhibitors of serine hydrolases,2s3 e.g., diethyl(4-nitrophenyl) phosphate, inactivate the five carboxylesterasesamidases rapidly and irreversibly.5,‘5 The organophosphorus diesters, bis(Cnitropheny1) phosphate and bis(Ccyanopheny1) phosphate,15 act similarly, but in contrast to the well known toxic inhibitors of serine ‘j E. Brandt, E. Heymann, and R. Mentlein, Biochem.

Pharmacol.

29, 1927 (1980).

344

ENZYME

PREPARATIONS

t461

hydrolases, the diesters are rather specific for liver carboxylesterasesamidases and exhibit low toxicity. l6 Bis(Ccyanopheny1) phosphate shows a preference for the pZ 5.6 esterase.‘j SpeciJicity and Physical Properties. Some data on the specificity of the five carboxylesterases/amidases are compiled in Table IV. A broader review also has been published.2 See Ref. 4 for the specificity of the dipeptidyl peptidase. Table V summarizes some physical properties of the six purified hydrolases. The pZ 5.6, pZ 6.0, and pZ 6.2 carboxylesterases show differing peptide maps after cleavage with trypsin or CNBr. Carboxylesterase pZ 6.4 is a glycosylated variant of carboxylesterase pZ 6.2. All esterases except those with pZ 5.2 and pZ 6.4 are essentially free of bound carbohydrates (R. Mentlein and E. Heymann, unpublished). K. Krisch, H. Biich, and W. Buzello, Biochem. Pharmacol.

I8 E. Heymann,

[46] Microsomal By

THOMAS

M.

Epoxide Hydrolase PHILIP

GUENTHNER,

18,801 (1969).

BENTLEY,

and

FRANZ

OESCH

Microsomal epoxide hydrolase (EC 3.3.2.3) catalyzes the conversion of epoxides to glycols. Because many epoxides formed from exogenous compounds are potent electrophiles, capable of covalent interaction with cellular molecules, the enzyme represents a key step in the detoxication of reactive intermediates. ‘d-5 Although found primarily in the microsomal fraction, the enzyme is also present in lesser amounts in other cell membranes.‘j The microsomal enzyme should not be confused with another epoxide hydrolase activity, found primarily in the cytosolic fraction, which differs greatly from membrane-bound enzyme in substrate specificity7-s and immunological properties. lo As with other membrane* F. Oesch,

Xenobiotica

3, 305 (1973).

2 F. Oesch, Prog. Drug Metab. 3, 253 (1979). 3 A. Y. H. Lu and G. Miwa, Annu. Rev. Phprmacol. Toxicol. 20, 513 (1980). 4 T. M. Guenthner and F. Oesch, in “Polycyclic Hydrocarbons and Cancer” (H. V. Gelboin and P. 0. P. Ts’o, eds.), Vol. 3. Academic press, New York 1981. 5 D. M. Jerina, P. M. Dansette, A. Y. H. Lu, and W. Levin, Mol. Pharmacol. 13,342 (1977). B P. Stasiecki, F. Oesch, G. Bruder, E. Jarasch, and W. W. Franke, Eur. J. Cell Biol. 21,79 (1980).

’ K. Ota and B. D. Hammock, Science 207, 479 (1980). 8 S. H. Mumby and B. D. Hammock, Pestic., Biochem. Physiol. 9 F. Oesch

and M. Golan,

lo T M. Guenthner, (1981).

METHODS

Cancer

Left.

11, 274 (1979).

9, 169 (1980).

B. D. Hammock, U. Vogel, and F. Oesch, J. Biol.

IN ENZYMOLOGY,

VOL.

77

Chem.

256,

3163

Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O-12-181977-9