305

Biochimica et Biophysics Acta, 617 (1980) 305-317 @ Elsevier/North-Holland Biomedical Press

BBA 57506

PURIFICATION OF RAT LIVER LYSOSOMAL CHOLESTERYL ESTER HYDROLASE

WILLIAM J. BROWN * and DEMETRIOS S. SGOLJTAS **

Emory University School of Medicine, Department of Pathology and Laboratory Medicine, Atlanta, GA 30322 (U.S.A.)

(Received April 23rd, 1979)

Key words: Cholesteryl ester hydrolase; Acid lipase; (Rat liver)

Summary

An acidic cholesteryl ester hydrolase (EC 3.1.1.13) from rat liver lysosomes was purified approximately 120-fold with 5% recovery of the original homo- genate activity. The sequential steps were: digitonin solubilization, agarose gel filtration, DEAE-agarose and CM-agarose column chromatography. The enzyme was at least 90% pure as judged by polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. It exhibited a molec- ular weight of about 60 000 as determined by sodium dodecyl sulfate polyacryl- amide electrophoresis and gel filtration. The soluble enzyme required substrate which was incorporated into phospholipid vehicles for optimal activity. On the contrary, aggregates of the enzyme required a substrate preparation that involved the direct addition of cholesteryl ester in acetone. The enzyme also catalyzed the hydrolysis of emulsions of triacylglycerol. The ratio of the two activities remained almost constant during purification suggesting that the two activities (EC 3.1.1.13 and EC 3.1.1.3, respectively) may be the result of the broad specificity of one enzyme. The effects of some inhibitors and some properties of the enzyme have been studied and discussed.

Introduction

Recent studies have demonstrated that the liver contains several cholesteryl ester hydrolase (EC 3.1.1.13) capable of hydrolyzing endogenous and exogenous cholesteryl esters. A ‘soluble’ cholesteryl ester hydrolase has been characterized

* Present address: Veterans Administration Hospital, Asheville, NC 28805. U.S.A.

* * To whom reprint requests should be addressed.

306

and partially purified by Deykin and Goodman [l]. The same authors have also described a microsomal cholesteryl ester hydrolase from rat liver. Riddle et al. [2] reported an acidic activity towards cholesteryl esters which was associated with plasma membranes. A lysosomal cholesteryl esterase from rat liver was first reported by Stoffel and Greten [3] and further studied by Nilsson et al. [ 41. Lysosomal cholesteryl ester hydrolase activity of human liver and of calf liver has been described by Stokke [ 5,6]. These activities have been studied primarily in crude preparations and some properties of the enzyme have been reported.

The availability of a stable purified cholesteryl esterase should facilitate continued studies on the substrate specificity, and cofactor requirements of the enzyme as well as comparison studies of lysosomal cholesteryl esterase with other lipolytic enzymes. The purpose of the present investigation is to report the purification and some characteristics of the lysosomal cholesteryl ester hydrolase from rat liver. Some of this work has been presented in abstract form

171.

Materials and Methods

Materials [1,2-3H]Cholesterol (spec. act. 52 Ci/mmol) and acylglycerol tri-[1-14C]-

oleate (spec. act. 65 mCi/mmol) were purchased from New England Nuclear (Boston, MA, U.S.A.). Nonradioactive acylglycerol trioleate, cholesteryl oleate, cholesterol and phosphatidylcholine (egg yolk lecithin) were obtained from Sig- ma Chemical Company (St. Louis, MO, U.S.A.). Other chemicals were obtained from the following sources: Digitonin from ICN-Life Sciences Group, (Nutri- tional Biochemicals, Cleveland, OH, U.S.A.); ‘Triton X-100 from Packard, (Downers Grove, IL, U.S.A.); bovine serum albumin, essentially free of fatty acids from Pentex (Kankakee, IL, U.S.A.); Nembutal from Abbott Laboratories (North Chicago, IL, U.S.A.); Sephadex G-200, Blue Dextran 2000, and standards for molecular weight determinations from Pharmacia (Piscataway, NJ, U.S.A.). Agarose gels (Bio-Gel A, DEAE Bio-Gel A and CM Bio-Gel A) from Bio-Rad Laboratories, (Richmond, CA, U.S.A.). All other chemicals were reagent grade or better.

Preparation 0 f lysosomes Male Sprague-Dawley rats weighing 200-250 g maintained on Purina

Laboratory Chow and water ad libitum were fasted overnight before they were sacrificed. To prepare the liver lysosomal fraction, a modification of the proce- dure of Teng and Kaplan [8] was followed. 5-6 animals were anesthesized by Nembutal (4-5 mg/lOO g body weight) which was injected intraperitoneally. The animals were killed by bleeding through puncture of the abdominal aorta. The chest was opened and the liver was cleared of remaining blood by perfusing in situ with 0.23 M sucrose (pH 7.4) until fully blanched. The liver was cut into pieces and homogenized in 8 ~01s. of 0.25 M sucrose, 1 mM EDTA (pH 7.4), using a glass-Teflon pestle homogenizer (Arthur H. Thomas, Philadelphia, PA, U.S.A.). ‘The pestle was moved through one up-down cycle six times. The homogenate was centrifuged at 1000 Xg for 10 min and the pellet was

307

discarded. The supematant was centrifuged at 2600 Xg for 30 min and the pellet (mitochondria) Was separated. The 2600 X g supematant was recentri- fuged at 12 000 X g for 30 min. The obtained pellet (light mitochondria) and its brown layer was manually homogenized in sucrose solution with an all glass tissue homogenizer and the suspension was centrifuged immediately at 12 000 X g for 20 min. The pellet (lysosome-enriched pellet) was further disrupted in order to release the latent lysosomal activities. In some experi- ments the two 12 000 X g supematants were combined and centrifuged at 100 000 Xg for 60 min in the preparation of microsomes and cytoplasmic extract (100 000 X g supematant).

Solubilization and purification of activity The following techniques for uncovering latent lysosomal activity were used:

(1) The lysosome-enriched pellets were suspended in 0.1 M NaCl, 0.01 M Tris- HCl buffer (pH 7.4), vigorously homogenized in an all glass apparatus and subjected to freeze-thawing using a dry ice-acetone bath and a 37°C water bath for 10 successive cycles. (2) Portions (0.5 ml) of a suspension of lysosomes (10 mg of protein/ml), in cold (2”(Z), 0.05 M Tris-maleate buffer (pH 7.4), were sonicated for three 1-min periods employing a Branson Sonifier Cell Disruption 200 (Branson Instr., Danbury, CT) ultrasonic disintergrator equipped with the standard microtip. (3) The washed lysosomes were suspended in Triton X-100, 0.1% and 0.5%, respectively, 0.01 M phosphate buffer (pH 7.4), and sonically disrupted. (4) The lysosomal pellet was homogenized in 0.25 M sucrose with an all glass tissue homogenizer and cold digitonin solution was added to the suspension to concentration of 0.5%, and the mixture was stirred at 4°C for 90 min. After each treatment the suspension was centrifuged at 50 000 X g for 30 min, the supematant was aspirated and used for experiments and for chromatography.

All chromatographic procedures were carried out at 4°C. The enzyme solu- tion from the clear supematant after the digitonin treatment solubilization step was applied to a 2.6 X 90 cm column of Bio-Gel A (1.5 m Agarose Gel, lOO- 200 mesh), previously equilibrated with 0.05 M Tris-HCl buffer, pH 8.3. The column was developed with the same buffer at a flow rate of 50 ml/h and approx. 120 fractions (5 ml each) were collected. Only those fractions showing high activities were combined and used in the subsequent experiments. In selected experiments, a 2.6 X 80 cm column of Sephadex G-200 previously equilibrated with 0.05 M Tris-HCl buffer, pH 8.3 was employed. The enzyme- containing fractions were passed through an ultrafiltration cell using a PM-10 filter membrane (Amicon Corporation, Lexington, MA, U.S.A.). The residue concentrated to 5 ml was applied to a 2 X 10 cm column, DEAE Bio-Gel A, 100-200 mesh, previously equilibrated with 0.05 M Tris-HCl buffer, pH 8.3. The column was developed by gradient elution from 0 to 0.25 M NaCl in the Tris-HCl buffer. Fractions of 3 ml were collected and those showing cholesteryl ester hydrolase activity were combined. The volume was reduced to about 10 ml by ultrafiltration (PM-10 membrane) and the pH was adjusted to 5.4 with cold 10% acetic acid. The enzymatic preparation was then placed on a 0.9 X 10 cm column packed with CM Bio-Gel A, 100-200 ,mesh, previously equilibrated with 0.05 M phosphate buffer, pH 5.4. After application of the

308

sample, the column was washed with about 4 bed volumes of buffer used for equilibration. Then, it was developed by gradient elution with a 0 to 0.25 M NaCl in the phosphate buffer. Fractions of 3 ml were collected.

Protein concentration in column fractions was determined by measuring the absorbance at 280 nm. Otherwise, protein was determined according to Lowry et al. [9], using bovine serum albumin as a standard.

Cholesteryl ester hydrolase assay The incubation mixture, usually in a final volume 3 ml, contained 0.15 M

acetate buffer (pH 4.8), 0.1 ml of a suitably diluted tissue fraction or enzymic preparation and 0.1 ml of substrate preparation. The latter was prepared following the method of Brecher et al. [lo] and liposomes were made from 60 mg of lecithin and 600 ~-lg [ 1,2-3H]cholesteryl oleate (8.0 &i). [ 1,2-3H]- Cholesteryl oleate was synthesized and purified as previously described [ 111. Incubations were carried out routinely at 37°C for 30 min with constant shaking. The reaction was terminated by the addition of 6 ml of chloroform/ methanol (2 : 1, v/v) containing unlabeled cholesterol and cholesteryl oleate. After the addition of 1.2 ml of 0.02% CaCl,, the tubes were shaken and centri- fuged at 3000 X g for 10 min. The lower phase was transferred to conical tubes, evaporated under vacua, redissolved in chloroform and a small aliquot applied on Silica gel G plates. The plates were developed in petroleum ether/diethyl ether/glacial acetic acid (95 : 5 : 1, v/v), the lipid classes were isolated and the radioactivity was determined by scintillation counting. In selected experiments an alternate procedure for measuring cholesteryl ester hydrolysis was used in which the labeled ester was introduced from an acetone solution [1] directly into the tissue fraction. For assays at different pH, the range of pH values between 3.0 and 8.0 was covered by 0.1 M citrate-O.2 M phosphate buffers. A standard microassay was essentially the same as above except that the reaction volume was 0.5 ml.

Triacylglycerol hydrolase assay For this assay acylglycerol tri[l-14C]oleate was used as substrate. The sub-

strate (5 mM) was emulsified in 1% gum arabic by ultrasonication for 1 min. The incubation mixture contained 1.0 pmol of radioactive acylglycerol tri- [l-14C]oleate (0.6 PCi), 5 pmol CaClz, 25 pmol of acetate buffer, pH 4.8 and enzyme preparation in a total volume of 0.5 ml. The reaction was started by the addition of substrate and allowed to proceed at 37°C for 30 min, with con- stant shaking. The reaction was terminated by the addition of Dole’s extraction medium [12] containing 8 * lo-’ M non-radioactive oleic acid as a carrier. The radioactive oleic acid produced was isolated and determined as described previously [ 131.

Marker enzyme assays Succinic dehydrogenase, a marker enzyme for mitochondria, was determined

according to Pennington [ 141. Glucose-6-phosphatase, a microsomal marker, was determined according to Hubscher and West [15]. 5’-Nucleotidase, a plasma membrane marker, was assayed according to Emmelot et al. [16]. Inorganic phosphorus from glucose-6-phosphatase and 5’-nucleotidase assays

309

was quantitated by the method of Bartlett [17]. Acid phosphatase was deter- mined by measuring the hydrolysis of fl-glycerophosphate as described by Berthet and DeDuve [ 181, except that the incubation medium was buffered with 0.1 M acetate buffer, pH 4.8.

Es terase assay Esterase was assayed with p-nitrophenylacetate as substrate as described by

Egeh-ud and Olivecrona [ 193, except that the incubation medium was buffered with 0.1 M acetate buffer, pH 4.8. Cloroform solutions of p-nitrophenyl esters were evaporated to dryness with a stream of nitrogen. The compounds (25 flol/ml) were then dispersed by heating to 60°C in 80 mg/ml Triton X-100. The release of products was measured as the increase in absorbance at 412 nm relative to a control reaction mixture which contained no enzyme.

Phospholipase assay The assay for phosphatidyl choline phospholipase A was modified from that

of Waite and van Deenen [ 201. The substrate suspension contained 0.6 mM phos- phatidylcholine, 50 mM sodium acetate buffer, pH 4.8, 1 mM CaCl, and 20 mg/ml Triton X-100.

Polyacrylamide gel electrophoresis and molecular weight determination Polyacrylamide gels (7.5%) were run at pH 8.9 according to Maurer [21],

however, stacking and spacer gels were not used. The gel dimensions were 5 mm diameter X 6 cm. The protein bands were stained with Amido Black. Gels were also run in the presence of SDS-polyacrylamide for molecular weight determination. The method of Weber and Osborn [22] was used for 7.5% acryl- amide gels except that persulfate was used at half the specified concentrations. Samples were prepared by incubating 100 ,ug of protein with 100 /JJ of 10 mM phosphate, pH 7, 1% SDS, 1% mercaptoethanol, in a boiling water bath for 2 min. Protein (approx. 20 H) was applied to each gel and a current of 8 mA/ tube was used during electrophoresis. Aldolase (158 000), bovine serum albumin (68 000), ovalbumin (43 000), subunit of aldolase (40 000), chymo- trypsinogen A (25 000) and ribonuclease A (13 700) were used as molecular weight standards. Protein bands were visualized by Coomassie blue staining [23]. Molecular weight was also determined using a 2.2 X 90 cm column of Sephadex G-200 equilibrated with 0.05 M Tris-HCl buffer pH 8.3. Blue dextran was used for the determination of void volume. The column was calibrated with the same standard proteins which were used for SDS-polyacrylamide gel electrophoresis.

Isoelectric focusing Thin layer polyacrylamide gel analytical electrofocusing was performed by

using a LKB 2117-101 kit containing ampholine carrier ampholytes in the range of 3.5-9.5. Samples were dialyzed extensively against distilled water, lyophihzed, dissolved in 5% sucrose, 1% ampholytes and applied to the top of the gel (anode). The anode buffer was 0.4% triethanolamine and the cathode buffer was 0.4% H,SO,.

310

Results

In preliminary studies, whole homogenate, disrupted lysosomal suspensions and solubilized fractions were incubated at pH values varying from 3 to 8. In aggreement with previous results [4,24,25] all preparations hydrolyzed [ 1,2-3H]cholesteryl oleate which was incorporated in egg yolk lecithin vesicles at an optimum pH between 4.5 and 5. At pH 4.8, hydrolysis proceeded at a linear rate for time periods up to 60 min when cholesteryl ester concentration was 50 ~01 and the incubations (3 ml) did not exceed 2, 1, 0.3 and 0.1 mg protein for whole homogenate, lysosomal suspension, solubilized lysosomal fraction, and purified enzymic preparation, respectively. The effect of substrate concentration was studied, and it was shown that for 30 min incubation, hydrolysis was linear with respect to substrate concentration at levels below 200 pmol. At higher concentrations the activity plateaued.

The distribution of cholesteryl ester activity among different subcellular fractions from rat liver was compared with that of marker enzymes (Table I). This comparison was made by a quantitative estimation of the relative specific activity in each sub-fraction isolated by differential centrifugation as described in Experimental procedures.

The relative specific activity of acid cholesteryl ester hydrolase clearly paralleled that of acid phosphatase, the enzyme which has been widely used as a lysosomal marker enzyme. When the enzymic preparation was tested towards triacylglycerol at acidic pH the results paralleled the distribution of acid phos- phatase. These data are consistent with the results of other authors who have convincingly shown that acidic lipolytic activities are associated with lysosomes or light mitochondria enriched in lysosomes [ 261.

Table II shows results from the extraction methods which were used in order to release the cholesteryl ester hydrolase activity from the lysosomal prepara- tion. The extracted activity was determined from the activity in the supema- tant fraction divided by that of the corresponding suspension. The results show that only 45% of the activity was extracted from the lysosomal particles by freezing and thawing. The same percent activity was extracted by sonication, although the amount of protein in the supernatant was higher, suggesting that some inactivation of the enzyme might have occurred during sonication. More activity was extracted when the particles were treated with 0.1% Triton X-100 and even more with 0.5% Triton X-100. More protein was extracted with the latter treatment, however, and the overall specific activity was lower indicating that Triton X-100 at higher concentration interfered with the assay. Treatment with digitonin yielded almost 90% of the activity in the soluble fraction. On the other hand, total protein was lower than that obtained with 0.5% Triton X-100 but higher than that obtained with 0.1% Triton X-100. Overall, the cholesteryl ester hydrolytic activity was better extracted with digitonin and hence the latter method was routinely used in the present work. The solubil- ized preparation contained other hydrolases, a few of which were assayed. Thus an extract which contained acidic cholesteryl ester hydrolase with a specific activity of 13.8 nmol per min per mg protein also had the following specific activities; acidic lipase, 20.7 nmol per min per mg protein; acidic phospholipase 63 nmol per min per mg protein; esterase 140 and 110 nmol per min per mg

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TABLE II

EXTRACTION OF CHOLESTERYL ESTER HYDROLASE ACTIVITY FROM RAT LIVER LYSO-

SOMES

AIiquots containing an average of 100 mg of protein were suspended in 6 ml of distilled water or extract-

ing solution at 4’C. Portions (5 ml) of the total suspension were then centrifuged at 5000 X g for 30 min

and activities and protein concentration in the supernatant fluid in the initial suspension were measured.

Each value is the average * S.D. of four separate experiments.

Method of extraction Fraction Protein (mg) Activity *

Freezing and thawing Supernatant

Suspension

Sonication Supernatant

Suspension

0.1% Triton Supernatant

Suspension

0.5% Triton Supernatant

Suspension

0.5% Digitonin supematant

Suspension

52.2 k 6.7

93.5 f 10.1

63.6 * 8.1

98.7 + 12.6

50.5 t 8.4

96.8 * 11.5

72.8 .k 9.6

92.4 f 10.8

71.0 i 8.5

98.8 + 10.1

6.9 k 0.8

15.3 * 1.6

5.1 * 0.9

11.6 k 1.8

11.2 + 1.6

51.1 Il.9

6.0 ? 0.9

9.3 ? 1.6

13.9 ?- 1.8

16.0 + 2.1

* Activity in firno1 of product formed per min per mg Protein multiplied by mg of protein.

protein for p-nitrophenyl myristate and p-nitrophenyl acetate, respectively. A typical elution pattern from the Bio-Gel A column is shown in Fig. 1. The

cholesteryl ester hydrolase activity was found to emerge as a large peak follow- ing the second protein peak. An approximately 12-fold enrichment was achieved by this step. Specific enzymic activity increased about 7-fold as compared with that in the homogenate. Lipolytic activity towards triacyl- glycerol substrate followed the same protein peak with cholesteryl ester hydrolase. When [ 1,2-3H]cholesteryl oleate was presented in acetone almost all of the hydrolytic activity was found to emerge from the column as a single peak with the void volume. In results not shown, the activity of the Triton X-100 treated lysosomal particles was found to emerge as a single peak with the void volume. In one experiment this activity was rechromatographed on a

FRACTION NO.

Fig. 1. Bio-Gel A filtration of digitonin treated cholesteryl ester hydrolase from rat liver lysosomes. Details of the procedure and assay of enzyme activity are described in the test.

313

_

FRACTION NO

Fig. 2. Elution profile from chromatography on DEAE Bio-Gel A after Bio-Gel A filtration of cholesteryl ester hydrolase.

Sephadex G-200 column and the protein was recovered in the void volume again.

Fig. 2 shows the elution pattern from the DEAE Bio-Gel A column. A single peak with cholesteryl ester hydrolase activity was eluted by the gradient at a molarity of about 0.1 M NaCl. Total enrichment was 16-fold.

Fig. 3 shows the elution patterns from CM Bio-Gel A column. The activity was eluted with the second of the two peaks. The active fractions were combined and constituted the purified enzyme preparation. The whole purifi- cation process is summarized in Table III. The purified enzyme was at least 90% pure as judged by photodensitometry of gels from polyacrylamide gel electrophoresis. Also, a single major band was obtained on SDS-polyacrylamide gel electrophoresis after the final state of purification. For molecular weight determinations, comparison with marker proteins was performed and the mobility of the band indicated a molecular weight of 60 000 2 2000. The molecular weight was also estimated by gel filtration on Sephadex G-200. The elution volume corresponded to that expected for a protein of about 58 000 +

10 20 30 40

FRACTION NO.

Fig, 3, Elution profile from chromatography on CM Bio-Gel A.

NaCl

CM)

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TABLE III

STAGES IN PURIFICATION OF LYSOSOMAL CHOLESTERYL ESTER HYDROLASE FROM RAT

LIVER

Specific activity is expressed as the /.mml [3Hlcholesterol released per min per mg protein. Incubation of

100 pm01 [1.2-3Hlcholesteryl oleate contained 2, 0.2 and 0.1 mg protein of the homogenate, the soluble

l~sosomes and the chromatographic fractions, respectively. -

Total Specific Total Purification Yield

protein (mg) activity activity fold

Homogenate 3585 2.1 10416 1 100

Soluble 460 13.8 6041 5 58

Bio-Gel A 178 20.0 3541 I 34

DEAE-Agarose 44 43 1895 16 18.2

CM-Agarose 1.5 316 485 117 4.9

3000 and thus confirmed the measurement made by SDS-polyacrylamide electrophoresis. Isoelectrofocusing gave one main band at pH 6.1 and two weak bands at lower pHs. Because of the small amount of protein isolated no attempt was made to extract the bands and -define activities. As a result, the precise nature of the relationship of the bands remains unknown.

The soluble fraction of rat liver homogenate, starting material for the isola- tion of acid cholesteryl e&erase, contained a large percentage of the acid lyso- somal triacylglyceryl lipase. In three experiments the two acid activities were measured during purification and the ratio of cholesteryl ester hydrolase to triacylglycerol hydrolase activity remained nearly constant throughout the purification (Table IV), suggesting that both activities were catalyzed by a single protein. Furthermore, cholesteryl ester hydrolase activity in the final purification stage was high (310 nmol per min per mg protein) for p-nitro- phenylmyristate and very low (18 nmol per min per mg protein) for p-nitro- phenylactate strongly suggesting that these result8 were typical of a true lipase [24,25].

The effect of several compound8 on the activity of purified acid cholesteryl ester hydrolase is shown in Table V. The addition of 1 mM of divalent metal

TABLE IV

COMPARISON OF CHOLESTERYL ESTER HYDROLASE AND TRIACYLGLYCEROL LIPASE

ACTIVITY FROM RAT LIVER LYSOSOMES

Units of cholesteryl ester hydrolase are as the number of gmol released per min per mg of protein. Units

of lipase activity are the number of &unol of fatty acid released per min per mg of Protein.

Specific activity

Sterol ester hydrolase Lipax Ratio (lipase/hydrolase)

Homogenate 2.7 3.8 1.42

Soluble 13.8 20.7 1.56

Bio-Gel A 20.0 39.6 1.98

DEAE Bio-Gel A 43 108.2 2.51

CM Bio-Gel A 316 408.5 1:29

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TABLE V

EFFECT OF VARIOUS SUBSTANCES ON CHOLESTERYL ESTER HYDROLYSIS

Assays contained 0.1 mg protein of the purified preparation and 100 pm01 cholesteryl oleate.

Compound Concentration Purified enzyme

hydrolysis (% of control)

CaClz

M&O4

5.5-Ditbiobis-

(2-nitrobenzoic acid)

N-Ethylmaleimide

p-Chloromercuribenzoate

Protamine sulfate

1mM 91

10 mM 6

1mM 92

5mM 56

1mM 50

5mM 35

10 mM 20

1mM 92

5mM 78

3mM 28

1mM 24

500 mg/ml 82

ions had little effect on the enzyme activity. The enzyme, however, was completely inhibited at 10 mM concentration of CaCl,. The purified enzyme was sensitive to NaF; it was 50% inhibited at 1 mM concentration of NaF and 80% inhibited at 10 mM concentration. Incubation of the enzyme with sulf- hydryl reagents, e.g. p-chloromercuribenzoate and N-ethylmaleimide produced approximately 75% inhibition suggesting the presence of sulfhydryl groups. However, another sulfhydryl reagent, 5,5dithiobis-(2nitrobenzoic acid), had less effect at either 1 or 5 mM concentration. The inhibition of the enzyme by protamine sulfate indicated that the enzyme had a low pl and therefore formed a complex with the highly basic protamine sulfate denying the active site of the enzyme access to the substrate.

In our experiments two types of cholesteryl ester hydrolase activity were seen: one toward liposomal substrates and another toward microcrystallic substrates (solution in acetone). The greatest activity with liposomal substrates was found in the supernatant after digitonin extraction. Less than 30% of the total activity remained in the pellet. There was negligible activity in the agarose void volume peak with most of the enzyme activity eluting in later, lower molecular weight fractions. This indicated an activity associated with solubil- ized protein. On the other hand, the greatest activity with acetone solubilized substrates was found in the pellet remaining after digitonin extraction and in the void volume peak eluted from the agarose gel filtration column. This indicated an activity associated with insoluble particles and large aggregates. In the final purified state the enzyme was active only towards liposomal sub- strates. The purified enzyme was stable at 4°C for several days. It lost 50% of its activity after a period of 2 weeks. Owing to the presence of proteolytic enzymes in the original preparation, enzyme preparations before the DEAE Bio-Gel A and CM Bio-Gel A steps were labile and had to be processed immediately. Pretreatment of purified enzyme at 60 or 160°C for 2 or 3 min caused complete loss of lipolytic activity towards cholesteryl esters.

316

Discussion

This paper reports an approximate 120-fold purification of rat liver lyso- somal cholesteryl ester hydrolase. The final preparation was stable at 4°C for a period of several days and it gave a single protein band on polyacrylamide and SDS-polyacrylamide gel electrophoresis. Isoelectric focusing, however, demon- strated some heterogeneity within the expected activity. The molecular weight of acid cholesteryl ester hydrolase by gel filtration was in agreement with the band of protein found on SDS-polyacrylamide gel electrophoresis, approxi- mately 60 000.

A lipolytic enzyme of similar size has been obtained from rat liver lysosomes in purified form by Teng and Kaplan [7]. The enzyme, however, was highly specific for triacylglycerols and had only a trace of residual activity for choles- teryl oleate. Furthermore, it required lipid for elution from chromatographic materials and for activation. At present, it is not known whether the two preparations represent identical enzyme proteins, or isoenzymes with similar active sites. In future experiments we will attempt to answer this question.

The release of latent enzyme from the lysosomal membranes was most effec- tively accomplished by digitonin treatment. In our hands, cholesteryl ester activity could not be released satisfactorily from the membranes without resorting to detergent treatment. Triton X-100 which has been extensively used in other procedures caused aggregation of the enzyme and the resulting species was excluded from a Sephadex G-200 suggesting a molecular weight of over 10’. The question may arise whether the higher molecular weight form is a distinct molecular species or a weight artifact from the association of the enzyme with particulate material from the agarose gel. A similar situation has been reported for triacylglycerol lipase in adipose tissue by Claycomb and Kils- heimer [27]. In that tissue also it has not been clearly shown, however, whether the two forms represented one enzyme or one form was an artifact of the isolation procedure. It is of interest to note, however, that the formation of high molecular weight aggregates was essential to the expression of the catalytic activity towards the microcrystallic form but not the liposomal form.

Presenting substrates in various modes in studies of lipolytic enzymes has been a common practice. In the current study, we used routinely the substrate system described by Brecher et al. [lo]. Those phospholipid vesicles have been shown before [lo] to consist predominantly of liposomes and to be the suit- able substrate for the assay of enzyme systems isolated from rat liver lyso- somes. Cholesteryl oleate incorporated in liposomes was the favored substrate throughout the purification process. This strongly suggested a lipase-like character for the enzyme with a requirement for an interface for a full expres- sion of its activity. This was further confirmed when it was shown that the enzyme in its purified form hydrolyzed miscellar p-phenylmyristate but not soluble p-phenylacetate.

Similarities between cholesteryl ester hydrolase and triacylglycerol lipase reported here, make it less likely that two completely different proteins are responsible for these enzymic activities. This assumption would require that cholesteryl ester hydrolase cochromatographed with triacylglycerol lipase through all systems. Or, that the contaminated enzyme had an extremely high

317

specific activity contributing little protein in the protein compositional anal- ysis. Because of the small amount of protein isolated at the present study no attempt was made to apply other criteria such as inhibitory activities or immunochemical properties in order to answer this question.

A more plausible hypothesis is that the two activities may be the result of the broad specificity of one enzyme, a triacylglycerol lipase-cholesteryl esterase complex with the two activities acting in concert and binding as one unit. This is reminiscent of the association of the two activities observed previously in adipose tissue [27]. Still an alternate explanation might be that the specificity attributed to the lipolytic activities may be highly dependent upon the physico- chemical nature of the substrate. The role of the enzyme in vivo as either a triacylglycerol lipase or cholesteryl esterase may be determined by the environ- ment of the lipid at or near the surface of the enzyme. In this regard, the work of Okeida and Fujii [28] with rat liver lipase and cholesteryl esterase is of interest. They found an interconversion of triacylglycerol lipase and a choles- teryl esterase from rat liver upon addition of lipids. In future studies we plan to employ well characterized lipids and study changes in the activity of the cholesteryl ester hydrolase.

Acknowledgements

The authors wish to acknowledge the excellent technical assistance Miss Mary Davis and Mrs. Fania Szlam. This work was supported by U.S. Public Health Service research grant HL 139800 and in part by RR 5364.

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