THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed In U. S. A. Val. 258,. No. 19, Issue of October 10, pp. 12043-12050.1983

Hydrolysis of Triolein in Phospholipid Vesicles and Microemulsions by a Purified Rat Liver Acid Lipase*

(Received for publication, March 4, 1983)

Robert E. Burrier and Peter Brecher From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 021 18

An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified prepa- ration using ethylene glycol and Triton X-100. A mo- lecular weight of 67,000-69,000 was determined in- dependently using density gradient centrifugation, so- dium dodecyl sulfate-polyacrylamide gel electropho- resis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiola- beled triolein into either unilamellar vesicles or mi- croemulsions. Substrates were prepared by cosonicat- ing aqueous dispersions of lecithin and triolein. For- mation of vesicles or emulsions depended on the rela- tive amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:l and 26:l (1ecithin:triolein) and the microemulsion preparation at a molar ratio of 1:l. The substrate particles were of similar size (220-250 A) as deter- mined by Bio-Gel A- 15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was sim- ilar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater V,,, val- ues (0-0.6 pmol/min/mg), and V,,, for the emulsion was 3.1 pmol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent in- hibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both prepa- rations.

Acid lipase activity has been localized in lysosomal fractions obtained from several cell types (1-9). Although the purifi- cation of an acid lipase from several mammalian sources has been reported, extensive characterization of the purified en- zyme has been difficult due to limited yields and the relative instability of the purified or partially purified enzyme (2, 7, 9). A recently reported procedure for the purification of an acid lipase from human liver by Warner et al. (8) circumvented extensive subcellular fractionation by solubilization with Tri- ton X-100, and stabilization of the enzyme was accomplished using ethylene glycol. The purified enzyme was shown to hydrolyze cholesteryl esters and triacylglycerols when these lipids were assayed as particles described as emulsions stabi- lized in Zwittergent 3-14 (8).

* This work was supported by United States Public Health Service National Institutes of Health Grant HL 12869. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Because of the insolubility of triacylglycerol and cholesteryl ester in aqueous systems, the substrate preparations used to assay for acid lipase activity have often been complex and poorly defined containing various detergents, proteins, or emulsifiers. In previous studies we have shown that choles- teryl esters or triacylglycerols could be incorporated into unilamellar lecithin vesicles which were stable and effective substrates for an acid lipase from liver and aortic tissue (10, 11). Similar preparations have subsequently been used by other investigators (6, 7, 12, 13).

A lysosomal acid lipase is believed to be responsible for the hydrolysis of triacylglycerols and cholesteryl esters contained within lipoprotein particles that enter the lysosomal system by endocytotic processes (14). Because lipoprotein particles have physical properties that would characterize them as microemulsions (15) a model substrate suitable for examining the action of an acid lipase on lipoproteins would be a stable microemulsion. Procedures for preparing stable microemul- sions of phospholipid and cholesteryl ester have recently been described (16). In this study we report the purification of an acid lipase from rat liver by a modification of the procedure described by Warner et al. (8) and describe the use of triolein- containing phospholipid vesicles and microemulsions as model substrates for this enzyme.

EXPERIMENTAL PROCEDURES

Materials-The following radioisotopes were obtained from New England Nuclear: tri[l-14C]oleoyl glycerol (99.8 mCi/mmol), dipal- mitoyl ph~sphatidyl[rnethyl-~H]choline (27 Ci/mmol), dipalmitoyl[Z- palmit0yl-9,1O-~H]phosphatidyl~holine (60 Ci/mmol), [9,10-3H]oleic acid (7.5 Ci/mmol), [phen~l-~]Triton X-100 (1.58 mCi/mg), bovine serum albumin [rnethyl-14C], (13.2 pCi/mg), y-globulin [methyl-14C], (8.7 pCi/mg), and lactoalbumin A [rnethyl-’*C] (21.9 pCi/mg). Seph- adex G-150, ConA-Sepharose and blue dextran 2000 were purchased from Pharmacia Fine Chemicals. Bio-Gel A-15m was purchased from Bio-Rad. N-Tetradecyl-N’,N’-dimethyl-3-ammonio-l-propane sul- fonate (Zwittergent 3-14) was obtained from Calbiochem-Behring, and egg yolk phosphatidylcholine (Grade 1) was purchased from Lipid Products, Surrey, U.K. Liquiscint is a product of National Diagnos- tics, Sommerville, NJ, and Intralipid (10%) was obtained from Cutter Medical Laboratories, Berkeley, CA. 4-Methylumbelliferyl palmitate and 4-methylumbelliferone were purchased from Sigma. Carbowax PEG-20,000 was purchased from Fisher, and all other chemicals were of the highest reagent grade available. The water used was deionized and distilled in glass to assure purity.

Purification of Rat Liuer Acid Lipase-The purification scheme used in this study for rat liver acid lipase was a modification of the procedure of Warner et al. (8) for the isolation of acid lipase from human liver. Purification was monitored using a fluorometric assay (17). Male Sprague-Dawley rats (250-400 g, Charles River Breeding Laboratories, Wilmington, MA) were killed by guillotine. The livers (75 g wet weight) were rapidly excised, placed into ice-cold buffer I (0.25 M sucrose, 0.01 M Tris, pH 7.4, and 1 mM EDTA) and minced. The tissue was homogenized in 4 volumes of buffer I using 3 passes of a Teflon-glass homogenizer with a clearance of 0.025 inch, and the procedure was repeated using a pestle allowing only 0.012 inch clear- ance. Unless otherwise noted, all the following manipulations were

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12044 Acid Lipase Action on Vesicles and Microemulsions

performed at 4 "C. The homogenate was centrifuged at 1600 X g for 10 min and the pellet was discarded. A lysosomal fraction was obtained by centrifuging the supernatant at 14,000 X g for 30 min, and the resulting pellet was resuspended in 160 ml of buffer I1 (1% Triton X-100, 10 mM sodium acetate, pH 5.0, 1 mM 2-mercaptoeth- anol, 1 mM MnC12, and 1 mM CaC12) by homogenizing for 3 min in a Waring blender. This dispersion was then centrifuged at 130,000 X g for 30 min in a Beckman Ti 60 rotor and the supernatant used for further purification.

A column of ConA-Sepharose (1.6 X 8 cm) was pre-equilibrated in buffer 11. The supernatant was applied at a rate of 1 ml/min. After the solution was passed completely over the column once, the effluent was recirculated over the column bed overnight with the aid of a peristaltic pump. The column was then washed with 150 ml of buffer 111 (0.5% Triton X-100, 33% ethylene glycol, 0.5 M NaCl, 10 mM 2- mercaptoethanol, 20 mM sodium acetate, pH 5.0, 1 mM MnC12, and 1 mM CaC12) followed by an additional 100 ml of buffer IV (0.5% Triton X-100, 33% ethylene glycol, 10 mM 2-mercaptoethanol, and 20 mM sodium acetate, pH 5.0). The column bed containing the absorbed enzyme was then removed from the column and the enzyme eluted batchwise at 25 "C with four 40-ml aliquots of a solution containing all the components of buffer IV plus 0.5 M a-methylmannoside, pH 3.75. The ConA-Sepharose was pelleted by brief centrifugation (5 min at 1600 X g) and after each successive extraction the supernatants were combined and stored a t -20 "C.

The combined supernatant was concentrated 10-fold by dialysis against 1.5 liters of 30% Carbowax PEG 20,000 at 4 "C for approxi- mately 30 h. Four-ml aliquots were then applied to a Sephadex G- 150 column (2.6 X 94 cm), pre-equilibrated in buffer IV but containing 0.1% Triton X-100, and eluted by ascending chromatography with the equilibration buffer a t a flow rate of 10 ml/h. Fractions containing enzymatic activity were pooled and stored at -20 "C. Calibration of the Sephadex G-150 column was performed using blue dextran 2000, 0.1 pCi of 3H20, and 0.1 pCi each of the 14C-methylated proteins y - globulin (MF = 150,000), bovine serum albumin (Mr = 68,000) and lactoalbumin A (M, = 18,400).

Analytical Methods-Protein was determined by the method of Lowry et al. (18) using bovine serum albumin as standard. Samples containing ethylene glycol and Triton X-100 were dialyzed exten- sively against distilled water prior to assay. In many cases addition of the phenol reagent caused the appearance of a flocculent material which was removed by brief centrifugation. Triacylglycerols were determined by the method of Fletcher (19) and phospholipids were assayed by the method of Bartlett (20). For polyacrylamide gel electrophoresis, the system described by Laemmli (21) was used with a stacking gel of 3.75% acrylamide and 0.1% N,N-methylenebisacry- lamide and a separating gel of 10% acrylamide and 0.26% N,N- methylenebisacrylamide. After electrophoresis the gel was stained with Coomassie brilliant blue R-250 using the technique of Weber and Osborn (22). Protein standards run with each gel included albu- min ( M , = 68,000), ovalbumin ( M , = 43,000), chymotrypsinogen (Mr = 25,000), and ribonuclease ( M , = 13,700).

Sucrose density gradient ultracentrifugation was performed by layering 0.2-ml samples onto 4.0 ml of a 10-30% continuous sucrose gradient containing 10 mM sodium acetate, pH 5.0. When designated, the gradients also contained 0.1% Triton X-100. Samples of the enzyme were dialyzed against 10 mM acetate buffer (pH 5.0) contain- ing 0.1% Triton X-100 prior to application. Gradients containing 0.1 pCi each of the I4C-methylated proteins y-globulin and bovine serum albumin ( M , of 150,000 and 68,000, respectively) were also centrifuged as standards. Centrifugation was performed for 16 h at 4 "C at 50,000 rpm using a Beckman SW 60 Ti rotor. Fractions (0.2 ml) were collected from the bottom of the tubes and analyzed for radioactivity or enzymatic activity using the fluorogenic assay. The sucrose con- centrations of the fractions were measured with a hand refractometer.

Preparation and Characterization of Substrates-Vesicles and mi- croemulsions were prepared by cosonicating aqueous dispersions of egg yolk lecithin and triolein. The designated amounts of labeled and unlabeled lipids were dissolved in ch1oroform:methanol (21, v/v), evaporated under a stream of nitrogen, and then lyophilized. The dried lipids were routinely resuspended in 7 ml of a solution contain- ing 0.1 M NaCl, 0.01 M Tris, pH 7.4, and 0.02% sodium azide. All vesicle preparations were prepared such that the final phospholipid concentration was 38 mM (assumed M , of 787) and contained 0.35 pCi/ml of [3H]lecithin. The amount of triolein added was varied to obtain appropriate molar ratios of phospholipid to triolein, but the amount of ['4C]triolein added was always 1.5 pCi/ml. The microe- mulsion was prepared by mixing equimolar amounts of egg yolk

lecithin and triolein so that after resuspension in aqueous buffer the concentration of each lipid was 14.1 mM. These suspensions routinely contained 0.35 pCi/ml of [3H]lecithin and 0.5-2.0 pCi/ml of ["C] triolein.

The resulting suspensions were then sonicated continuously under a nitrogen atmosphere with a Branson W-350 sonicator fitted with a standard 0.5-inch horn at a power setting of approximately 3.5. The temperature within the glass-jacketed sonication cell was maintained between 37 and 42 "C. For vesicle preparation, sonication time was 20-30 min and the microemulsion preparation was sonicated for 1 h. The resulting opalescent mixtures were then centrifuged in a Beck- man 50 Ti rotor at 40,000 rpm for 60 min at 5 "C. From the ultracen- trifuge tube, the upper 1 ml was removed and 4 ml of the remaining infranatant was then transferred to another tube and centrifuged a t 42,000 rpm in a Beckman SW 60 Ti rotor for 16 h at 5 "C. The tubes were then fractionated and aliquots assayed chemically or isotopically for phospholipid or triacylglycerol. For subsequent use as substrates, vesicles were obtained in the lower 1.0-ml fraction and the microe- mulsion was collected in the upper 1-ml fraction of the appropriate centrifuge tube.

To characterize the vesicle or emulsion preparation, 0.2-ml aliquots were applied to a Bio-Gel A-15m column (2.6 X 37 cm) that was pre- equilibrated with a solution containing 0.1 M NaCl, 0.01 M Tris, pH 7.4, and 0.02% sodium azide. Samples were eluted with the equilibra- tion buffer in an ascending manner using a peristaltic pump at a flow rate of 0.40 ml/min. The void volume and the total volume were determined by blue dextran 2000 and tritiated water, respectively. Chromatography was perfomed a t ambient temperatures (22-25 "C) . Eluted fractions were then analyzed for radioactivity with Liquiscint as the scintillation cocktail in a Packard Tricarb 300 CD programmed to calculate the disintegrations per min for each isotope.

To isolate lipoproteins, freshly collected human serum was adjusted to 0.1% EDTA and fractionated by sequential ultracentrifugation in a Beckman 60 Ti rotor. VLDL' (d < 1.006 g/ml) and LDL (d = 1.006- 1.063 g/ml) fractions were obtained as described previously (23) and dialyzed extensively against 0.15 M NaCl and 1 mM EDTA, pH 7.4, prior to use.

Assay for Lipase Actiuity-Assays using vesicles or microemulsions as substrates were performed similarly. Incubation tubes (13 X 100 mm test tubes) contained 50 mM sodium acetate, pH 4.4, 2.5 mM 2- mercaptoethanol, 0.5 mM EDTA, the designated amount of labeled substrate in volumes not exceeding 100 pl, and 10 pl of the purified enzyme preparation, all in a final volume of 250 pl. Twenty-min incubations a t 37 "C were routinely performed. Following incubation the reaction was terminated by the addition of 3.5 ml of ben- zene:chloroform:methanol (1.00.5:1.2, v/v/v) containing unlabeled oleic acid (0.1 mM) as carrier. An 0.6-ml aliquot of 0.3 M NaOH was then added, and the resulting suspension was mixed for 15 s followed by centrifugation at 1000 X g for 10 min to separate the phases. The free fatty acid was then determined by counting a 1-ml aliquot in 10 ml of Liquiscint in a scintillation counter. Under these conditions 89% of the free fatty acid partitioned into the upper phase. All measurements were made in duplicate.

Assays performed to determine the products of the reaction were scaled to 1 ml. Incubations were terminated by addition of 20 volumes of ch1oroform:methanol (21, v/v), followed by 5 volumes of 0.9% NaCl and 1 mM H2S04. The lower phase was removed, dried under a stream of nitrogen, and spotted on a thin layer plate (Silica Gel G) in a minimal amount of solvent. The plate was chromatographed in a solvent system of hexane:diethyl ether:acetic acid (70305, v/v/v). Regions corresponding to appropriate standards were scraped into scintillation vials and counted in 5 ml of Liquiscint.

During the purification procedures, activity was measured by a fluorometric assay using 4-methylumbelliferyl palmitate as substrate exactly as described by Warner et al. (17). Fluorescence was measured using a Turner fluorometer (model 111) with excitation at 360 nm and emission at 450 nm with 4-methylumbelliferone as standard.

RESULTS

Purification of Acid Lipase-Table I summarizes results from a typical purification procedure. Activity was measured using a fluorometric assay employing surfactant-stabilized emulsions of 4-methylumbelliferyl palmitate as substrate.

The abbreviations used are: VLDL, very low density lipoprotein; LDL, low density lipoprotein.

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Acid Lipase Action on Vesicles and Microemulsions

This assay was reliable and reproducible at all purification stages and was effective in the presence of the relatively high concentrations of Triton X-100 and ethylene glycol required during the purification and subsequent storage. The minimum specific activity reported is 2000-3000 nmol/min/mg which is an order of magnitude lower than that reported by Warner et al. (8) for the human liver lipase but was comparable to that reported by other workers for acid lipase obtained from several sources (1,24). The enzyme was purified at least 80-fold from the lysosomal fraction. This value was not precise due to difficulty in obtaining an accurate protein determination, presumably due to interference by Triton X-100 which gave high blanks and which we were unable to remove by extensive dialysis, surfactant exchange, absorption to polystyrene beads, or incubation with phospholipid vesicles. This problem of obtaining accurate protein values on the Sephadex G-150 eluate was encountered with the Lowry procedure (18), Coo- massie blue binding (25), fluorescamine labeling (26), and absorbance techniques. Attempts to further purify the acid lipase by carboxymethylcellulose, Sephadex LH-20, octyl- Sepharose, or Sephacryl S-200 column chromatography pro- duced no appreciable increase in specific activity but losses in total activity were observed.

Fig. 1 shows the elution profile of the enzyme from the Sephadex G-150 column which was calibrated with radiola- beled proteins. The estimated molecular weight was 69,000

TABLE 1 Purification of acid lipase from rat liver

Purification was initiated using 75 g of rat liver (wet weight). One unit of activity corresponds to 1 nmol of 4-methylumbelliferone formed per min.

step Protein

tion Volume concentra- Activity specific Yield tivity

38

W

W 24

cn 0 W

30

Y

9 18

d 12

6

7s 4.8s

2 6 10 14 18 22 I " I

FRACTION

FIG. 2. Effect of Triton X-100 on the sedimentation of acid lipase in sucrose density gradients. Samples of the purified acid lipase were dialyzed against 10 mM acetate buffer, pH 5.0, containing 0.1% Triton X-100. Aliquots (0.2 ml) were applied onto 10-30% sucrose gradients either lacking or containing 0.1% Triton X-100. Following centrifugation, the activity in each fraction was measured using 4-methylumbelliferyl palmitate as substrate. B, activity in gradients containing Triton X-100; 0, activity in gradients not con- taining Triton X-100; 0, per cent sucrose. Standards: y-globulin, 7 S albumin, 4.6 S.

ml m g / m l unitlml unitlmg 9% Lysosomal frac- 187 10.4 343 32.9 (100)

Triton X-100 ex- 183 6.5 420 64.6 115

ConA-Sepha- 167 0.29 278 958 70

Sephadex (2-150 294 0.018 48 2666 21.5

tion

tract

rose

l8kDa V+

1 1

12045

30

25 -? 3

c5

Y

" $ W

U 0

15 2 f-

10 0

a

s U W

5

A B C D " "I

38kDa

l3kDa

250 350 450

ELUANT (ml)

FIG. 1. Elution profile of acid lipase activity following gel filtration on Sephadex G-150. Column was pre-equilibrated and eluted using buffer IV but containing O.l%'Triton X-100 (see text). Activity was monitored using 4-methylumbelliferyl palmitate as sub- strate. The column was standardized using blue dextran 2000, VO; y- globulin, 150 kDa; albumin, 69 kDa; lactoalbumin A, 18 kDa; and 'H20, V,.

!BkDa

FIG. 3. SDS-polyacrylamide gel electrophoresis of acid li- pase at different stages of purification. Lane A, lysosomal frac- tion following solubilization with 0.1% Triton X-100. Lane E, fraction following elution from ConA-Sepharose. Lane C, fraction following gel filtration on Sephadex G-150. Lane D, standards: albumin ( M , = 68,000); ovalbumin ( M , = 43,000); and chymotrypsinogen (M, = 25,000). For lanes A and B material was applied directly to the gel. For lane C the fraction was concentrated approximately 50-fold prior to application.

based on a K., of 0.42. An independent estimate of molecular weight of the pooled fractions from the Sephadex G-150 column was obtained by sucrose density gradient ultracentri- fugation (Fig. 2). When the gradient was run in the absence of Triton X-100, activity was not detected in any fraction even if Triton X-100 was added to the assay tubes subsequent to centrifugation. In the presence of 0.1% Triton X-100, activity was readily detected as a single peak sedimenting as a particle with a sedimentation coefficient of 4.5 S correspond- ing to a molecular weight of 67,000 Da, based on calibration with 14C-labeled albumin and y-globulin. Fig. 3 shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions from different stages of purification. Following

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12046 Acid Lipase Action on Vesicles and Microemulsions

gel filtration, a major band on the gel was observed corre- sponding to 69,000 Da. A minor band also was observed corresponding to 77,000 Da. Thus, chemical and enzymatic procedures suggest that rat liver acid lipase has a molecular weight between 67,000-70,000 Da and was approximately 85% pure at the final purification step. A minimum molecular weight of 29,000 for the human acid lipase was reported (8) while purified acid lipases from rat liver, human placenta, bovine thyroid, and human leukocyte have been reported ranging from 31,000-74,000 (1, 5, 7, 9, 24).

Preparation and Characterization of Lipid Substrates-The substrates used in this study were prepared by sonicating dispersions of lecithin and triolein of defined molar ratios between 70:l to 1:l (phospholipidtriolein), centrifuging the mixtures for 1 h at 150,000 X g to sediment titanium and float large emulsion particles, and then centrifuging at 200,000 X g for 16 h. The resulting preparations were characterized as either vesicles or microemulsions depending on the sedimen- tation or flotation following 16 h of ultracentrifugation. For all preparations, less than 1% of the total lipid sedimented during the first centrifugation for 1 h. When the initial molar ratio was 70:1, no lipid was detected at the air-water interface of the tube, but as the molar ratios were altered to increase the triolein content, progressively more lipid floated to the surface. At a molar ratio of 16:1, 16% of the triolein and 8% of the phospholipid were localized in a creamy layer at the surface, and for the preparation of molar ratio 1:1, about 36% of the triolein and 12% of the phospholipid floated.

Table I1 shows the distribution of labeled lipid in the ultracentrifuge tube following prolonged centrifugation (16 h a t 200,000 X g) of the infranatants obtained from the 1-h centrifugation of samples containing initial molar ratios of 70:1, 16:1, and 1:l. For the 70:l preparation (sample 1) over 65% of the total lipid sedimented to the lower part of the tube, and the remaining lipid was distributed throughout the tube. The molar ratio was essentially constant in all fractions. For the 16:l preparation (sample 2), again most of the lipid sedimented, but the molar ratio of the lower fraction was 27:l whereas the particles that did not sediment were enriched relative to triolein. About 9% of the triolein associated with a small amount of lecithin floated to the surface suggesting the presence of microemulsions in the mixture. When the 1:l mixture (sample 3) was centrifuged, about 60% of the lipid added to the tube floated to the surface with a molar ratio of 1:1 in that fraction. The lesser amounts of lipid distributed throughout the tube had molar ratios from 2.5 to 7.3. Chemical analysis for triacylglycerol and phospholipid was also per- formed on the fractions in the different tubes and the data were consistant with the values obtained based on specific

activities of the labeled lipids. The data indicated that under appropriate sonication conditions, starting mixtures of 701 to 16:l molar ratio produce mainly vesicles whereas microe- mulsions are formed when the starting molar ratio was 1:l.

The particles isolated by prolonged ultracentrifugation were also characterized by gel filtration (Fig. 4). The sedimenting fractions containing molar ratios of 70:l and 27:l eluted mainly as a single peak which was included in the column. The 70:l preparation eluted at a volume corresponding to that of unilamellar lecithin vesicles which are prepared in the absence of triolein, whereas the 27:l preparation eluted as a broader peak at a volume corresponding to slightly larger particles. The microemulsion prepared by flotation also was included in the column and eluted a t a volume similar to that of the 27:l vesicles. For all preparations, both the [3H]lecithin and ['4C]triolein coeluted in the same region, although the molar ratio differed somewhat in the ascending and descend- ing portion of the peaks suggesting slight heterogeneity within each of the preparations. In all cases, small amounts of labeled lipid were noted at the void volume, presumably due to aggre- gation of the vesicles or microemulsions. A small tritium peak was also found at the total volume, but chemical analysis indicated no phospholipid in this region, and the radioactivity was found to be an impurity in the [3H]lecithin.

Enzymatic Studies-The hydrolysis of triolein incorporated into either phospholipid vesicles (70:l molar ratio) or microe- mulsions under different assay conditions is shown in Fig. 5. Although the amount of fatty acid formed was consistently greater when microemulsions were used as substrate, no major differences between the two substrates were seen with respect to the effect of temperature or pH. Optimal activity was observed over a relatively broad temperature range between 37 and 50 "C. The pH optimum for both substrates was about 4.3 and activity was negligible at pH values above 6.5. Hy- drolysis increased with incubation time for at least 2 h with both substrates and was linear for about 30 min. The reaction rate was directly proportional to the amount of enzyme from 0-0.4 pg corresponding to 0-20 pl of the purified enzyme preparation. When more enzyme was added, the reaction rate decreased. This behavior was attributed to the presence of Triton X-100 in the enzyme preparation (see below). Based on the data in Fig. 5, assays were routinely performed at pH 4.4 for 20 min a t 37 "C, and 0.18 pg (10 p1) of enzyme was added.

Because Triton X-100 and ethylene glycol were present in the enzyme preparation, the effect of these substances on the standard assay was tested. Triton X-100 inhibited hydrolysis of both vesicles (85 p~ triolein) and microemulsions (500 pM triolein) in a dose-dependent manner. Fifty per cent inhibition

TABLE I1 Distribution of ['*C]triolein and PHllecithin contained in substrate preparations following prolonged

ultracentrifugation Substrate preparations were prepared by sonicating samples with initial molar ratios of 701 (Sample l ) , 16:l

(Sample a), and 1:1 (Sample 3). Following sonication each sample was centrifuged for 1 h a t 150,000 X g from which 4.0 ml of the infranatant were removed and centrifuged a t 200,000 X g for 16 h a t 5 "C. The tubes were fractionated and the radioactivity determined in each fraction. Data are expressed as the per cent of total radioactivity present in the samples centrifuged for 16 h. Molar ratios were calculated from specific activities.

[3H]Lecithin ["CITriolein Lecithin/triolein

Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3

% of total molar ratio

1 (TOP) 1.3 3.7 44.8 2.5 9.2 73.0 64 7 1.0 2 4.5 4.3 10.5 5.4 8.1 7.1 74 11 2.5 3 9.0 5.2 6.7 10.7 7.0 4.8 74 16 2.1 4 8.5 6.7 5.3 9.9 7.0 2.7 76 21 2.8 6 8.0 6.3 6.0 10.1 6.9 2.6 79 23 3.6 6 (Rottom) 67.1 67.3 22.6 58.2 58.8 4.9 79 27 7.3

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Acid Lipase Action on Vesicles and Microemulsions 12047

FRACTION

FIG. 4. Gel filtration of substrate preparations on Bio-Gel A-15 m. Aliquots (0.2 ml) of substrate preparations obtained follow- ing prolonged ultracentrifugation were applied to a column (2.6 X 37 cm), and gel filtration was performed as described under “Experimen- tal Procedures.” A, vesicles of molar ratio 701 (1ecithin:triolein) containing 412 and 420 dpm/pl of [3H]lecithin and [“Cltriolein, respectively. B, vesicles of molar ratio 27:l containing 2351 and 1263 dpm/Fl of [3H]lecithin and [14C]triolein, respectively. C, microemul- sions of molar ratio 1:1 containing 1000 and 1090 dpm/pl of [3H] lecithin and [‘4C]triolein, respectively. 4.0-ml fractions were collected and the data are expressed as dpm/0.4-ml aliquot counted.

was observed at concentrations of about 0.01% and 0.005% Triton X-100 for the vesicle and microemulsion, respectively. Ethylene glycol had no effect when added in amounts 5-fold greater than that normally included in the standard assay. Oleate, a reaction product, was added as the sodium salt and had no appreciable effect a t concentrations up to 250 p~ using either substrate.

To determine the reaction products, the reaction mixture was extracted by the procedure of Folch et al. (27) and the distribution of the labeled products determined by thin layer chromatography. When vesicles (70:l molar ratio) were used as substrates and incubation was for 45 min, 5% of the labeled

triolein was hydrolyzed and the molar distribution of fatty acid, mono-, and diglyceride was 3:2:1, respectively. Using the microemulsion as substrate under similar conditions, about 8% was hydrolyzed and the relative distribution of products was 5:3:2. Following prolonged incubation for 6 h at 37 “C, up to 14% of the triolein was hydrolyzed with proportionately more mono- than diglyceride. In all cases, the data indicated that monoglyceride was not hydrolyzed in the assay system and all the fatty acid formed was derived from di- or triolein degradation.

Incubations were also performed to determine the stability of the substrates following incubation at standardized condi- tions. Following the reaction, samples with either the 6 0 1 or 27:l vesicles or the 1:l microemulsion were applied to a Bio- Gel A-15m column and the elution profiles obtained were virtually identical with those shown in Fig. 4.

Phospholipase activity in the enzyme preparation was tested by preparing unilamellar vesicles that contained triti- ated lecithin labeled in either the choline or fatty acid moie- ties. These vesicles were incubated with the enzyme for pro- longed time periods up to 2 h under standardized conditions and the reaction mixtures analyzed for water-soluble products using the extraction described for the lipase assay or the procedure of Folch et at. (27). No detectable phospholipase activity was observed using vesicles lacking triolein or using vesicles containing triolein (70:l molar ratio).

Fig. 6A shows the effect of triolein concentration using vesicles (molar ratios of 60:1, 50:1, and 27:l and with the microemulsion (molar ratio 1:l) as substrate. Saturation was observed with each of the vesicle substrates but the maximal reaction rate was progressively greater as the vesicles were more enriched in triolein. A sigmoid shape was observed when vesicles were used as substrates (Fig. 6A). When using the microemulsion as substrate saturation was observed with no evidence of a sigmoid-shaped plot. The maximal activity was about 3.1 pmol/min/mg, clearly greater than that obtained for any of the vesicle preparations.

Fig. 6B shows a double reciprocal plot for the microemulsion both in the absence and presence of 10 pg of Triton X-100, an amount approximately equal to that contained in 10 pl of the purified enzyme preparation. Without the added deter- gent, a straight line was obtained giving values of 230 p~ and 3.1 pmol/min/mg for the apparent K,,, and V,,,,,, respectively. When Triton X-100 was added the plot was nonlinear at the lower substrate concentrations. Also shown in Fig. 6B is the data obtained using the larger emulsion particles (molar ratio 1:1.6) that were prepared by flotation from the 1-h centrifu- gation of the initial sonication mixture. This preparation has the same VmaX as the smaller emulsion although the apparent K,,, was greater (1800 pM).

Double reciprocal plots for the vesicle preparations are shown in Fig. 6C and nonlinearity was observed in all cases. This nonlinearity was even more obvious at substrate concen- trations lower than that shown in the figure and became still more pronounced if Triton X-100 were added to the reaction mixture (data not shown). The data in Fig. 6C is consistent with the presence of a substance in the enzyme preparation (Triton X-100) that interacts irreversibly with the substrate, producing nonlinearity when the ratio of detergent to sub- strate is high. Overestimation of a substrate (triolein) in a reaction would produce sigmoid velocity versus substrate plots and nonlinearity for double reciprocal plots (28, 29). Estima- tion of the amount of triolein removed from the substrate pool by Triton X-100 may be approximated by linearization of the double reciprocal plots shown following sequential subtraction of small (2 pM) quantities of substrate from each experiment. When correlation coefficients for each line were

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Acid Lipase Action on Vesicles and Microemulsions

FIG. 5. Characteristics of the as- say for acid lipase using either ves- icles (molar ratio 70:l) or microe- mulsions (molar ratio 1:l) as sub- strate. All incubations were performed at 37 "C for 20 min a t pH 4.4 with 0.18 pg of protein unless stated otherwise. A, effect of incubation temperature. Each assay tube contained either 12 p~ tri- olein as vesicles or 688 pM triolein as microemulsions. B, effect of pH. Each assay tube contained either 40 pM tri- olein as vesicles or 228 pM triolein as microemulsions. C, effect of incubation time. Each assay tube contained either 10 p~ triolein as vesicles or 1000 p M triolein as microemulsion. D, effect of enzyme concentration. Each assay tube contained either 24 pM triolein as vesi- cles or 1000 p~ triolein as microemul- sions. The enzyme preparation was as- sumed to contain 18 Fg/ml. Each point is the average of duplicate determina- tions. Similar data were obtained using vesicles of molar ratio 27:l.

FIG. 6. Effect of substrate concen- tration on acid lipase activity. A, varying amounts of either vesicles or mi- croemulsions were added to assay tubes and the reaction assayed under stand- ardized conditions. 0, vesicles with mo- lar ratio 601; A, vesicles with molar ratio of 50:l; A, vesicles with molar ratio of 26:l; 0, microemulsions of molar ratio 1:1. B, double reciprocal plots of kinetic data using the microemulsion as sub- strate. C, double reciprocal plot of kinetic data using vesicles as substrate.

0

DH

Triolein ( uM)

10 100 1000 10 100 1000 Neutral LDid ( us)

80 ID

1250

500 8 250

n P 4

< P

6 n B rn 0

B 1: 1.6 Emulslon

p 1:l plus Trlton

- 1: 1 Emulslon 4 LL - 0 5 10 . s E"

11s x 10-3(," )

FIG. 7. Effect of LDL, VLDL, and Intralipid addition on acid lipase activity. A, vesicles as substrate. All assay tubes con- tained vesicles of molar ratio 60:l at a final concentration of 28 pM triolein. Increasing amounts of either LDL (O), VLDL (A), or Intra- lipid (.) were added to the reaction mixture prior to incubation under standardized conditions. Amount of substance added was expressed as the total amount of neutral lipid (cholesteryl ester plus triglyceride) added per assay tube. Control value for vesicle-associated triolein hydrolysis was 0.155 pmol/min/mg. B, microemulsions as substrate. All assay tubes contained microemulsions (molar ratio 1:l) at a final concentration of 640 p~ triolein. Control value for microemulsion- associated triolein hydrolysis was 2.18 pmol/min/mg.

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Acid Lipase Action on Vesicles and Microemulsions 12049

optimized, V,,, for each vesicle type did not change signifi- cantly but the apparent K,,, values for all vesicles were ap- proximately 30 pM.

The effect of human VLDL and LDL on hydrolysis of both vesicles and microemulsions is shown in Fig. 7 . For both substrates LDL was a more effective inhibitor than VLDL when the data was expressed as the amount of neutral lipid (triglyceride plus cholesteryl ester) added. 50% inhibition was produced by approximately 5 and 100 pg of neutral lipid from LDL and VLDL, respectively. Inhibition also was produced by Intralipid, a commercially available triacylglycerol emul- sion.

DISCUSSION

Several lipases have been shown to hydrolyze neutral lipid contained in surface monolayers (30,31). These data strongly suggest that lipolysis occurs on the surface of biological emul- sions. Lipoprotein particles, which have the properties of microemulsions (15), are degraded intralysosomally, presum- ably by several enzymes including the acid lipase. To assess the mechanism by which the acid lipase degrades microemul- sions such as lipoproteins, we prepared model substrates of either microemulsions or vesicles containing neutral lipid. Sonication of mixtures of phospholipids and cholesteryl esters have been shown to generate vesicles or microemulsions de- pending on the starting lipid ratio (16). Using a similar approach, we prepared vesicles and microemulsions contain- ing lecithin and triolein.

The maximum solubility of surface-oriented triolein in a vesicle was reported to be about 3-4 mol % based on NMR studies (32). Thus, the vesicles we prepared a t 4 mol % should have essentially all the triolein accessible to the surface of the particles and also contain a saturated surface of triolein. When the triolein content is increased to a molar ratio of 1:1, microemulsions are formed in preference to vesicles. Emul- sions of lecithin and triolein are reported to have a surface containing 2-5 mol % triolein (33). In order to compare the hydrolysis of vesicle- and microemulsion-contained triolein, great care was taken to characterize the substrate prepara- tions with respect to both size and composition. Theoretically the vesicle containing 4 mol % triolein and the microemulsion should have nearly identical surface compositions. Since the sizes of vesicle and microemulsion preparations are very sim- ilar, it is, therefore, possible to compare the hydrolysis on a per particle basis.

Comparisons between the hydrolysis of triolein contained in either the microemulsion or the vesicles showed similarities with respect to pH activity profiles and incubation tempera- ture, suggesting that the same enzyme acts on both substrates. The most striking difference between the two types of sub- strates related to the kinetic data obtained. The V,,, value obtained using the microemulsion or 1:1.6 emulsion was con- siderably greater than that obtained for any vesicle prepara- tion. Assuming that vesicles with 4 mol % triolein and the emulsions both have saturated surfaces with respect to tri- olein, the different V,,, values for both substrate types sug- gests that the concentration of triolein on the surface was not the sole limiting factor in determining the reaction rate.

The difference between the K, values observed between the 1:l microemulsion and the 1:1.6 emulsion again suggests that surface-oriented triolein is hydrolyzed and the greater K, for the 1:1.6 emulsion most likely reflects the relatively large amount of triolein contained in the core of these particles and, therefore, not directly accessible to the enzyme. Using the microemulsion as substrate, an apparent K, of about 230 FM was calculated based on the total triolein concentration. Assuming that the enzyme acts solely at the lipid-water

interface and that approximately 4% of the triolein in the microemulsion is localized on the interface, a K, value cor- rected for surface-oriented triolein of 10 p~ is obtained. Presumably triolein in vesicles is distributed randomly throughout the bilayer; thus 67% of the triolein in each preparation is situated in the outer leaflet at any time (34). Therefore, the apparent K, calculated for the different vesicle preparations (30 p ~ ) when corrected to account for triolein in the outer leaflet is about 20 pM. When corrected for surface- oriented substrate, apparent K, values between vesicles and microemulsions differ by a factor of 2. This difference, al- though based on several approximations, may reflect different affinities of the enzyme for either particle surface.

The action of lipases on an interface has been reviewed recently by Verger (35). A proposed mechanism for hydrolysis involves binding of the lipase to the surface of a particle, followed by localization of the substrate to the active site. Upon hydrolysis of one substrate molecule the enzyme is free to either dissociate from the particle or continue to hydrolyze substrate on the same particle. Should dissociation be less likely, the diffusion of substrate within the particle to the active site may become important in determining the maximal velocity. The greater V,,, in microemulsions compared to vesicles could be due to a more rapid movement of core triolein to the surface in microemulsions than the lateral movement of triolein in the vesicles.

When comparisons were made between different vesicle preparations the V,,, increased proportionally as the relative amount of triolein in the vesicle increased. This is consistent with an increasing concentration of substrate available on the surface of the particle, which would lessen the lateral diffusion time between enzyme and substrate, possibly explaining the increasing V,,,.

Criticism concerning the use of emulsions for studying the properties of a lipase were based on size heterogeneity and the lack of detailed compositional data for previously used emulsion preparations (35, 36). The microemulsions used in this study have not been previously described and were char- acterized with respect to size and composition. Using such preparations it was possible to show that surface-oriented triolein was hydrolyzed by the acid lipase and that the core of an emulsion can effect the rate of neutral lipid hydrolysis. Such microemulsions are amenable to more detailed studies on the physical properties of surface and core phases using other techniques. The effectiveness of the microemulsion as a substrate for acid lipase, the similarities to lipoproteins with respect to size, and the demonstrated ability of lipoproteins to inhibit lipase activity toward the microemulsion suggest that these synthetically prepared particles may be useful in elucidating the mechanism by which acid lipase acts on nat- urally occurring microemulsions such as lipoproteins.

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R E Burrier and P Brecherrat liver acid lipase.

Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified

1983, 258:12043-12050.J. Biol. Chem. 

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