Partial purification and characterization of a microsomal carboxylesterase specific for salicylate esters from guinea-pig liver

138

Biochirnica et Biophysica Acta, 785 (1984) 138-147 Elsevier

BBA31828

PARTIAL PURIFICATION AND CHARACTERIZATION OF A M I C R O S O M A L CARBOXYLESTERASE SPECIFIC FOR SALICYLATE ESTERS FROM GUINEA-PIG LIVER KENNETH N. WHITE and DEREK B. HOPE

Department of Pharmacology, University of Oxford, South Parks Road, Oxford, OXI 3QT ( U.K.) (Received June 2nd, 1983) (Revised manuscript received October 25th, 1983)

Key words: Carboxylesterase,"Aspirin hydrolase; Molecular weight," Substrate specificity; (Guinea-pig liver microsome)

Studies on liver carboxylesterases have predominantly involved the use of uncharged ester and amide substrates to monitor activity. A microsomal carboxylesterase (EC 3.1.1.1) from guinea-pig liver microsomes has been identified which specifically hydrolyses aspirin (White, K.N. and Hope, D.B. (1981) Biochem. J. 197, 771-773), a substrate which is negatively charged at physiological pH, and this work describes its partial purification and characterization. The enzyme is monomeric, it has a molecular weight of approx. 55 000 and is very sensitive to inhibition by the carboxylesterase inhibitor bis(4-nitrophenyl)phosphate. Although it could not be completely separated from contaminating carboxylesterases, substrate specificity was investigated using the negatively charged esters of salicylic acid. The enzyme is not specific for the acetyl ester of salicylic acid, aspirin, but hydrolyses the longer chain esters more rapidly, with the highest limax for the n-octanoyl ester. The enzyme was subject to substrate inhibition which increased with increasing chain length of the fatty acid on the ester, and approached 100% inhibition at concentrations of substrate below critical micellar concentrations.

Introduction

Most studies on liver carboxylesterases which have included purification of enzyme, have used esters and amides which are uncharged at physiological pH as substrates to monitor enzyme activity. Recent work has involved characterization of a chloramphenicol hydrolase from guinea-pig liver [1], of methyl butyrate, acetanilide and 4nitrophenylacetate hydrolytic activities in rat liver [2] and of 1-mono-oleoylglycerol hydrolysis in rat liver [3]. It has been recognised since the studies of Bernhammer and Krisch [4] with a purified pig liver acetanilide hydrolase, that those carboxylesterases that have been isolated using uncharged substrates to measure activity, show little or no Abbreviation: BNPP, bis(4-nitrophenyl)phosphate. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

tendency to hydrolyse charged substrates. A comparison of turnover numbers of acetanilide hydrolase purified by Bernhammer and Krisch [4], showed that the uncharged substrate acetanilide was hydrolysed 1000 times faster than the negatively charged ester N-acetyl-4-aminobenzoic acid. Pig liver carboxylesterase purified by Levy and Ocken [5] could hydrolyse only one ester linkage of uncharged dicarboxylic diesters including dimethyl malonate, succinate, glutarate and adipate. The negatively charged monomethyl ester of succinic acid was neither hydrolysed by the enzyme nor inhibited it. Exactly the same behaviour was demonstrated by Wynne et al. [6] for a purified bovine liver carboxylesterase. Hofstee [7-9] has used straight chain fatty acid esters of 3-hydroxybenzoic acid, which are negatively charged at physiological pH, to investigate

139

carboxylesterase activities in liver. The studies concentrated on the kinetic characteristics of these carboxylesterases in crude extracts of liver, and indicated that, in several species, a single enzyme was responsible for this activity, although a purification was not undertaken. Reports from this laboratory concerning hydrolytic activities towards aspirin, a negatively charged carboxylester, in guinea-pig liver, have described the identification of a microsomal carboxylesterase as being the predominant aspirin hydrolysing activity of particulate cell fractions from this tissue [10,11]. A second, minor aspirin hydrolysing activity was identified in this tissue which was located exclusively in the cytosol fraction recovered from liver homogenates. This report describes the partial purification and characterization of the microsomal aspirin hydrolase in guinea-pig liver. Part of this work has already been published in abstract form [11]. Materials and Methods

The assays of aspirin, thioaspirin (acetyl-2mercaptobenzoic acid) and 1-naphthylacetate hydrolysis were carried out as described before [10]. Protein was assayed according to the method of Lowry et al. [12] using bovine serum albumin as a standard. Polyacrylamide gel electrophoresis was performed in slabs 18 x 14 cm of 7% polyacrylamide according to the method of Davis [13]. Carboxylesterase activities were stained with 5 mM 1-naphthylacetate in 0.1 M phosphate buffer pH 6.5, containing 20 mg Fast Blue RR (Sigma, Poole, U.K.) per 50 ml of buffer. Staining for aspirin hydrolysis was carried out as described previously [14]. SDS-polyacrylamide gel electrophoresis for routine analysis of enzyme preparations was carried out in 10% polyacrylamide slabs, 18 x 14 cm, according to the method of Weber et al. [15]. Subunit molecular weight was determined by electrophoresis according to the method of Laemmli [16] in 8% polyacrylamide slabs calibrated using marker proteins from a Sigma 6 kit (Sigma), comprising lysozyme (14300), lactoglobulin (18400), trypsinogen (24000), pepsin (34700), egg albumin (45 000) and bovine albumin (67 000). Proteins were

stained by incubating gels in a solution 0.25% (w/v) in Coomassie Blue R (Sigma, Poole, U.K.) in water (5 parts), acetic acid (1 part) and methanol (5 parts), for 45 min, followed by destaining in the same solution without dye.

Purification of aspirin hydrolase Freshly dissected livers from adult guinea-pigs of either sex were homogenised in 0.3 M sucrose in the proportion of 1 g wet weight tissue to 4 ml of sucrose, at 900 rpm (10 strokes) in a Potter-Elvejhem homogeniser. The homogenate was centrifuged at 10000 x g for 10 min, the supernatant retained and the sediment resuspended in 0.3 M sucrose to the initial volume of the homogenate, by homogenisation (10 strokes). After centrifugation at 10000 x g, for 10 min the washed sediment was discarded. The combined supernatants were centrifuged at 100000 x g for 30 min, and the microsomal pellet so obtained was resuspended by homogenisation in 25 mM Tris-HC1 (pH 8.0) (hereafter referred to as buffer), 1.0%' (w/v) in saponin (B.D.H. Chemicals, Poole, U.K.), to the original volume of the homogenate. The preparation was stirred at 0-4°C for 60 min and then centrifuged at 100 000 x g for 60 min. The supernatant, containing solubilised aspirin hydrolase, was fractionated with ammonium sulphate. The bulk of the aspirin hydrolysing activity was located in the 50-70% saturation fraction which was resuspended in, and dialysed against, buffer. The preparation was applied to a 90 x 5 cm column of Sephadex G-100 equilibrated with buffer, and was eluted with buffer at a flow rate of 22 ml. h -1. The aspirin hydrolase peak of the eluate was applied to a 30 x 2.2 cm column of DEAE-Sephadex A-50 equilibrated with buffer. The column was washed with 200 ml of buffer in 25 mM NaCI. The aspirin hydrolase was eluted with a linear gradient of NaC1 in buffer from 25 mM to 125 mM over 12 column volumes (1200 ml). Aspirin hydrolysing fractions of the eluate were pooled and dialysed by ultrafiltration on an Amicon 202 cell using a CM10 filter. When subjected to further purification, the pooled eluate was dialysed by ultrafiltration against 25 mM sodium phosphate (pH 7.0). The pH of the preparation was reduced to 5.5 by addition of 25 mM phosphoric acid, and applied to a 30 cm x 2.2 cm column of CM Sepharose CL-6B equilibrated

140 with 25 mM sodium phosphate buffer (pH 5.5). The aspirin hydrolase protein was eluted with a linear salt gradient of 0-125 mM NaC1 in 25 mM sodium phosphate buffer (pH 5.5).

Kinetic studies The esters of salicylic acid used in these studies were obtained as follows: aspirin was purchased from B.D.H.; n-butyryl salicylic acid was a gift from Dr. E. Mtiller, Karl Thomae GmbH, Biberach, F.R.G.; pivaloyl salicylic acid was a gift from Bayer GmbH. Oher esters of salicylic acid were prepared by acylation of salicylic acid neutralised with dimethylaniline. Salicylic acid (0.061 mol) was dissolved in dimethylaniline (0.122 mol) and cooled to - 1 0 ° C . The appropriate acid chloride (0.061 mol) was added in small portions over a period of 15 min, keeping the temperature below - 5 ° C . The reaction mixture remained at room temperature overnight and then shaken with 250 ml 2 M HC1 at 0°C. A crystalline product separated rapidly. After drying over CaC1 z in vacuo to constant weight, the material was recrystallised from petroleum ether (b.p. range 60-80°C). The yield of the crude material was approx. 90% of theory and upon recrystallisation recovery was approx. 60%. All assays were carried out in 100 mM sodium phosphate buffer (pH 8.0), 5 mM in 2-mercaptoethanol. Hydrolysis was assayed according to the method of Spenney [17], by continuous monitoring of salicylate release at 300 nm in a Pye Unicam SP 800 spectrophotometer. Enzyme and buffer were incubated at 37°C for at least 30 min prior to addition of substrate, also equilibrated at 37°C. After mixing of enzyme and substrate, the preparation was transferred to the cuvette in the spectrophotometer, also maintained at 37°C. Corrections were made for spontaneous hydrolysis of substrate. Kinetic data were analyzed by a method appropriate to the analysis of substrate inhibition described by Cleland [18]. The molecular weight of aspirin hydrolase was determined according to the method of Whitaker [19] by gel filtration. A column of Sephadex G-100 was calibrated with the globular proteins ribonuclease (13700), chymotrypsinogen (25000), ovalbumin (43000) and bovine albumin (67000). Elution volumes of proteins, Dextran Blue 2000

and aspirin hydrolase were determined as described previously [14]. The dependence of aspirin hydrolase activity on pH was measured using buffers made with 25 ml of 800 mM Tris, titrated to the required pH with a solution of glacial acetic acid and saturated succinic acid, 2:1 (w/v) and made up to 50 ml with water. Results

Purification of aspirin hydrolase Attempts to solubilise aspirin hydrolase from microsomal membranes by autolysis according to the method of Heymann et al. [20] were not successful (White, K.N. and Hope, D.B., unpublished data), and the use of a non-ionic detergent, saponin, was adopted, following the method of Mentlein et al. [2] to solubilise rat liver carboxylesterases. Following solubilisation, conventional procedures were used to purify aspirin hydrolase. Two carboxylesterase peaks were resolved by gel filtration, the bulk of the activity eluting as the high molecular weight form, and the aspirin hydrolase peak was approximately coincident with the low molecular weight carboxylesterase peak (Fig. 1). Fractionation of the aspirin hydrolase peak on DEAE-Sephadex A-50 separated two carboxylesterase peaks (Fig. 2), the minor one of which was conincident with the aspirin hydrolase. A small amount of a second aspirin hydrolysing peak was coincident with the main carboxylesterase peak, but accounted for less than 3% of the total activity recovered in this step. The purification data shown in Table I indicate that the ratio of aspirin to thioaspirin hydrolysing activity at each step of the purification remained constant, and was similar to the ratio of the aspirin and thioaspirin hydrolase peak heights obtained from gel filtration or ion-exchange chromatography (Figs. 1 and 2). This confirms that the two substrates are hydrolysed by the same enzyme as was earlier assumed when thioaspirin was used as a substrate to stain aspirin hydrolases in polyacrylamide gels [10]. Attempts to purify the DEAE aspirin hydrolase fraction further by gel filtration on Sephadex G-75, chromatography on hydroxyapatite or on CMSepharose CL-6B met with little success (White,

141

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142 TABLE 1 ASPIRIN HYDROLASE PURIFICATION DATA Specific activity of the fractions are expressed in /~mol.min 1.mg l, the total activities of the fractions in /~mol.min-I, and the yields of the fractions as a percentage of the total activity present in the microsomes. Substrate

Microsomes

Saponin solubilizate

SephadexG-100 gel filtration

DEAE-SephadexA-50 chromatography

CM-SepharoseCL-6B chromatography

0.249 726.8 100

0.306 558.0 1.23 77

2.181 441.2 4.02 61

11.310 116.5 45.46 16

0.905 2.58 3.64 0.5

0.071 208.2 100

0.074 135 1.04 65

0.477 96.9 6.69 46

2.741 28.3 38.44 14

0.232 0.66 3.25 0.3

1-Naphthylacetate Specific activity Total activity Purification Yield

0.312 910.6 100

0.361 658.9 1.16 72

0.516 104.5 1.66 11

8.58 8.84 27.53 0.97

2.53 7.2 8.12 0.60

Protein Content (mg. ml- 1) Total (mg)

2.54 292.1

2.05 182.5

1.51 20.3

0.10 10.3

0.048 2.85

Aspirin Specific activity Total activity Purification Yield Thioaspirin Specific activity Total activity Purification Yield

K.N. and Hope, D.B., unpublished data). The first two techniques did not resolve aspirin hydrolase from contaminating carboxylesterases, and the third resulted in a marked inactivation of the aspirin hydrolase although some purification was achieved. Electrophoretic analysis of the D E A E fraction showed that the aspirin hydrolase constituted the major protein of the preparation (Fig. 3a and c), but that the contaminating proteins represented high carboxylesterase activities (Fig. 3b). Treatment of this fraction with chromatograp h y on CM-Sepharose CL-6B results in a partial removal of contaminating carboxylesterases, so that a single band due to aspirin hydrolase was revealed by staining for protein (Fig. 3c) and for thioaspirin hydrolysing activity (Fig. 4), although staining for carboxylesterase activity is evidently more sensitive and shows that the contaminating activities were still present (Fig. 3b).

Molecular-weight determinations The molecular weight of aspirin hydrolase present in a saponin solubilisate of freshly prepared microsomes was found to be 54 300 + 900 (mean

+ S.D. for five measurements), by gel filtration. The subunit weight of a preparation purified up to and including D E A E c h r o m a t o g r a p h y was found to be 57100 + 1500 (n = 23) by SDS-polyacrylamide gel electrophoresis, indicating that aspirin hydrolase is monomeric. The p H dependency of aspirin hydrolase activity is shown in Fig. 5. The o p t i m u m lies in the range of p H 7.0-8.5, in c o m m o n with other carboxylesterases [21], although the cause of the unusual shape of the p H rate-profile is not understood. A single band of enzymic activity is present in polyacrylamide gels stained with thioaspirin at p H 5.5.

Kinetic studies The partially purified preparation of aspirin hydrolase obtained from D E A E - S e p h a d e x A-50 c h r o m a t o g r a p h y contained several contaminating carboxylesterases, for which reason kinetic studies were limited to members of the homologous series of esters of salicylic acid, which are assumed to be hydrolysed by the aspirin hydrolase only. The dependence of hydrolytic activity on substrate

143

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Fig. 3. Polyacrylamide slab gel electrophoresis of guinea-pig liver microsomal proteins at different stages of purification. Following the protocol described in the Methods section, samples were obtained at successive stages of purification, after (1) saponin solubilisation of microsomes, (2) Sephadex G-100 gel filtration (3) DEAE-Sephadex A-50 ion-exchange chromatography, (4) CM-Sepharose CL-6B ion-exchange chromatography. Gels were stained for (a) protein with Coomassie Blue R250, (b) carboxylesterase activity with 1-naphthyl-acetate, (c) protein after SDS-electrophoresis. 10

c o n c e n t r a t i o n for this series of esters is s h o w n in Fig. 6. T h e e n z y m e is n o t specific for aspirin, b u t h y d r o l y s e s the l o n g e r c h a i n esters o f salicylic a c i d

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0

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Fig. 5. Dependence of aspirin hydrolase activity on pH. Samples of enzymes were purified up to and including DEAE-Sephadex A-50 chromatography. Each point represents the mean_+ S.D. of five measurements at each pH, using a single batch of enzyme.

144 Hydrolytic activity p Mol.min71mg71

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more rapidly at least at the lower substrate concentrations tried. At higher concentrations of substrate, there is an increasing tendency of these esters to inhibit the enzyme as the chain length of the fatty acid on the ester increases. Thus, at 5 mM, n-decanoyl salicylic acid inhibited the en-

TABLE II

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KINETIC PARAMETERS OF SALICYLATE HYDROLYSED BY ASPIRIN HYDROLASE

1000 1 3 ° ° 1! ~

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Km (M×104)

I/max (#mol-min ].mg -1)

Aspirin n-Propionyl ester n-Butyryl ester n-Valeryl ester n-Hexanoyl ester n-Octanoyl ester n-Decanoyl ester

4.47 5.76 7.99 9.83 6.79 2.01 1.32

19.20 31.25 83.70 67.80 103.10 130.60 37.00

1

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145 zyme 99%, but no inhibition was observed with aspirin, n-propionyl- or n-butyryl-salicylic acids. The inhibition could not be attributed to formation of products since the enzyme was fully active in the presence of higher concentrations of product than were present when the enzyme was inhibited at high concentrations of substrate. The aspirin hydrolase showed no activity towards the pivaloyl(2,2-dimethylpropionyl) ester of salicylic acid, indicating a specificity of the enzyme for straight chain esters. The values of apparent K~ and VmaX derived from analysis of kinetic data are given in Table II. Although the apparent Vmax in the series of esters was greatest for n-octanoylsalicylic acid, the rate % Inhibition 100-

of hydrolysis of substrate at a concentration of 0.1 mM was greatest for n-decanoylsalicylic acid. The apparent inhibition constants were all greater than 6 mM, except for n-decanoylsalicylic acid, which had a value of 0.1 mM, as expected for the marked substrate inhibition shown by this compound. The sensitivity of aspirin hydrolase to inhibition by the organophosphate carboxylesterase inhibitor BNPP is illustrated by the dependence of extent of inhibition on concentration of inhibitor, shown in Fig. 8. An aliquot of the enzyme used in this study was made 10 -6 M in BNPP, which inhibited the enzyme completely. No activity was recovered after dialysis of the inhibited enzyme against 25 mM Tris-HC1 (pH 8.0), 1 mM in EDTA, 0.1% (w/v) in 2-mercaptoethanol, for 3 days at 4°C, with four changes of buffer. A sample of untreated enzyme dialysed in the same way retained its activity. On this basis, the inhibition of aspirin hydrolase by BNPP was considered irreversible.

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Fig. 8. Inhibition of aspirin hydrolase by BNPP. Each point represents the mean + S,D. of five measurements using a single preparation of enzyme purified up to and including DEAESephadex A-50 chromatography. Inhibitor was incubated with enzyme for 15 rain at 37°C before remaining activity was measured.

The carboxylesterase activity of guinea-pig liver microsomes was found to occur mainly as the high molecular weight form of these enzymes, in agreement with earlier findings [1,22]. This enabled the bulk of this activity to be separated from aspirin hydrolase and other low molecular weight carboxylesterases by gel filtration. Attempts to purify the aspirin hydrolase further using conventional techniques met with only partial success. It was possible to remove non-carboxylesterase protein readily, but problems were encountered when attempts were made to separate aspirin hydrolase from low molecular weight carboxylesterases with similar physical properties. Aspirin hydrolase could be resolved from the bulk of the contaminating carboxylesterases by DEAE-Sephadex A-50 chromatography, but incompletely and with a low recovery of 26%. In contrast, Kuhn and Heymann [1] used the technique to good effect in the purification of chloramphenicol hydrolase from guinea-pig liver microsomes, with a yield of 85%. The aspirin hydrolysing peak from DEAE Sephadex A-50 chromatography consisted primarily of aspirin hydrolase protein, and the contaminating carboxylesterases could be partially removed by CM-Sepharose CL-6B chromatography, result-

146

ing in the detection of a single protein band by electrophoresis (Fig. 3a and c) due to aspirin hydrolase (Fig. 4). However, a massive loss (98%) of activity occurred during this step, even though 28% of the protein was recovered. Such behaviour has not been observed with carboxylesterases before, and carboxymethyl chromatography at low pH has been used to good effect by Zerner and co-workers [23-25] in the purification of carboxylesterases of livers from several species. The results of the purification study given in Table I show that the enzyme is present in significant quantities in guinea-pig liver, as it can be purified to a preparation that is predominantly aspirin hydrolase with only a 45-fold increase in specific activity, relative to that of the microsomal fraction. A similar conclusion was reached by Kuhn and Heymann [1] for the chloramphenicol hydrolase which was purified to more than 90% purity and 23-fold, starting from microsomes. The ability of aspirin hydrolase to hydrolyse preferentially longer straight chain esters of salicylic acid than aspirin is similar to the behaviour of a high molecular weight carboxylesterase purified by Inoue et al. [26] from human liver. The human enzyme hydrolysed the octanoyl ester of salicylic acid the fastest, out of all the members of the homologous series of straight chain fatty acid esters of salicylic acid from the acetyl to the nonanoyl ester. However, rates were determined at a single, unspecified, substrate concentration, and no kinetic parameters were determined. 2-Naphthylacetate was used to monitor activity of the enzyme during its purification, so it is not clear whether this enzyme is the only one responsible for aspirin hydrolysis in human liver. A comparison of the kinetic parameters obtained for the salicylate esters and aspirin hydrolase with values tabulated by Heymann [21] for aliphatic uncharged carboxylesters as substrates for purified pig and rat liver carboxylesterases reveals similar specificities of the enzymes for their respective substrates [27-29]. One pig liver carboxylesterase showed greatest specificity for methyl octanoate amongst methyl esters of straight chain fatty acids with a K m of 0.4 mM and Vmax of 565 t~mo1 • min- 1. mg- l, compared with 0.2 mM and 130 ~ m o l - m i n - I .mg ~ for the hydrolysis of n-octanoyl salicylic acid by aspirin hydrolase.

The substrate inhibition could not be attributed to formation of hydrolysis products of the salicylate esters, nor does it seem likely to be due to formation of micelles by substrate, shown by Hofstee [30] to be the reason for the substrate inhibition observed of a horse liver carboxylesterase and two hog pancreatic carboxylesterases by the n-octanoyt esters of 3- and 4-hydroxybenzoic acids. The n-octanoyl ester of 2-hydroxybenzoic acid (salicylic acid) was found to be largely unassociated at a concentration of 20 mM in 37.5 mM veronal buffer (pH 8.0), whereas the other two esters were largely micellar at this concentration. The microsomal aspirinase was found to be very sensitive to the carboxylesterase inhibitor BNPP, especially when compared with its cytoplasmic counterpart [14], which may be due to the negative charge on the inhibitor molecule at neutral pH. A similar correlation of avidity of an enzyme for a negatively charged substrate and inhibitor was found by Hattori et al. [31] who investigated the inhibition by BNPP of the hydrolytic activity of rat liver microsomes towards steroid acetates, aspirin and steroid hemisuccinate esters, which were inhibited for less than 20%, for 40% and for more than 90%, respectively by 10-5 M BNPP. Several features of the aspirin hydrolase described here which serve to distinguish it amongst carboxylesterases include: (a) its sensitivity to BNPP, (b) its ability to hydrolyse negatively charged substrates as avidly as other carboxylesterases hydrolyse their best substrates, (c) the poor yield from, or inactivation which occurs during ion-exchange chromatography, (d) the marked substrate inhibition, and to a lesser extent (e) it being a monomeric carboxylesterase. These features seem likely to have arisen by using a negatively charged carboxylester as substrate, and the enzyme may be related to a single carboxylesterase identified by Hofstee [7-9]] in livers from several species, which hydrolyses short or medium straight chain fatty acid esters of 3-hydroxybenzoic acid, and which were all low molecular weight carboxylesterases, except in the pig. The physiological role, if any, of aspirin hydrolase remains obscure, although the enzyme does share some features in common with lysophospholipase II isolated from bovine liver by De Jong

147

et al. [32]. This enzyme is monomeric, of molecular weight 60000, hydrolyses carboxylesters, was inhibited by BNPP and was located primarily in the microsomal fraction obtained from bovine liver homo~enates [33]. The phospholipid substrates for lysophospholipase II carry negative charge and were hydrolysed more avidly than either of the uncharged synthetic carboxylesters tried, tributyrin and 4-nitrophenylacetate. Lysophospholipids were shown not to be hydrolysed at all by purified preparations of liver carboxylesterases [2,32].

Acknowledgements This study was supported by a grant-in-aid from the Royal Society. During this work K.N.W. held a Medical Research Council studentship.

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