Comparison of the action of lipoprotein lipase on triacylglycerols and phospholipids when presented...

Eur .I Riochem 197, 315-321 (1991) (- FEBS 1991

001429569100240M

Comparison of the action of lipoprotein lipase on triacylglycerols and phospholipids when presented in mixed liposomes or in emulsion droplets Camilo ROJAS, Thomas OLIVECRONA and Gunilla BENGTSSON-OLIVECRONA Department of Medical Biochemistry and Biophysics, University of Umei, Sweden

(Received November 5, 1990) - EJB 90 1306

We have compared the action of lipoprotein lipase on liposomes of egg yolk phosphatidylcholine containing less than saturating amounts of trioleoylglycerol (< 3%) and emulsion droplets of the same lipids. The amounts of the two types of lipid particles (expressed in terms of phosphatidylcholine) needed to reach substrate saturation of the enzyme were similar, indicating similar binding of the lipase to these two lipid/water interfaces. With liposomes, as opposed to emulsion droplets, albumin was not necessary for continued hydrolysis of triacylglycerols, presumably because product fatty acids could be accommodated in the phospholipid bilayer. The maximal rate of trioleoylglycerol hydrolysis was more than 10-fold higher, and the ratio of trioleoylglycerol/phosphatidylcholine hydrolysis was more than 50-fold higher with the emulsion droplets. Qualitatively similar results were obtained with hepatic lipase, and a lipase from Pseudomonasflu0rescenc.e. The data suggest that the lipases remained at the interface for several catalytic cycles, and that a continued supply of substrate molecules to the active site favored triacylglycerol entry from the core of the lipid particle, rather than sliding in from the side through lateral diffusion in the surface layer.

The reactions catalyzed by lipases take place at lipid/water interfaces (Verger and deHaas, 1976; Brockman, 1984). The enzyme diffuses in solution until it binds to the lipid surface containing the substrate molecules. This binding may be as- sociated with conformational changes in the enzyme molecule. In the next step, a substrate molecule is aligned into the active site of the enzyme, and its ester bond is split. Finally, the products must leave the active site so that a new substrate molecule can enter. It is not known if this occurs by diffusion of the product molecules in the interface, or if the enzyme must jump to a new site on the same or on another lipid particle. Monolayers of substrate lipids at air/water interfaces have been used to study lipase reactions in detail (Verger and deHaas, 1976; Brockman, 1984). These studies have shown that the reaction is highly dependent on the surface composition and on the packing of lipid molecules, properties which have been summarized by the term ‘quality of the interface’ (Verger and deHaas, 1976).

Lipoprotein lipase and hepatic lipase are two related enzymes (Jackson, 1983; Persson et al., 1989) which act on plasma lipoproteins. These have cores of triacylglycerol and other hydrophobic lipids enveloped by a surface layer of phospholipids, cholesterol and specific apolipoproteins (Miller and Small, 1987). Studies with liposomes have shown that long-chain triacylglycerols can be solubilized in bilayers of phosphatidylcholine up to about 3 mo1/100 mol (Hamilton and Small, 1981; Hamilton, 1989), and a similar amount of triacylglycerol is presumably present in the surface layer of phospholipid-stabilized triacylglycerol emulsions, and in

Correspondence to G. Bengtsson-Olivecrona, Medicinsk Kemi 5, Umea Universitet, S-90187 Umei, Sweden

Abbreviufion. Apo, apolipoprotein. Enzyme. Lipoprotein lipase (EC 3.1 .I .34).

plasma lipoproteins (Miller and Small, 1987). It appears likely that the lipases act on the lipids contained in this surface layer. It is known that lipoprotein lipase and hepatic lipase hydrolyze both phospholipids and triacylglycerols in the plasma lipoproteins (Jackson, 1983; Eisenberg and Rachmilewitz, 1975) but what factors govern the relative activity against the two types of lipid molecules is not known.

In the present study, we have compared liposomes containing less than saturating amounts of triacylglycerols with emulsion particles made up of the same lipids, as substrates for lipoprotein lipase. We speculated that if the lipase reaction was solely dependent on the quality of the interface, then the liposomes and the emulsion particles would support similar reactions. The results show, however, that with emulsion droplets triacylglycerol hydrolysis is highly favored over phospholipid hydrolysis. In other words, the reaction depends not only on the surface composition, but also on what is below the surface. To study if this is unique for lipoprotein lipase, we have carried out similar experiments with the related hepatic lipase, and with an unrelated bacterial lipase.

EXPERIMENTAL PROCEDURES

Enzymes and activator

Lipoprotein lipase was isolated from bovine skimmed milk by chromatography on heparin-Sepharose (Bengtsson-Oli- vecrona and Olivecrona, 1985). Hepatic lipase was isolated from human post-heparin plasma also by chromatography on heparin-Sepharose (Zaidan et al., 1985). The resulting preparation is free from lipoprotein lipase but contains some other proteins. Therefore the activity is given by volume of enzyme solution, rather than by mass. Lipase from Pseudomonasfluo-

316

rescence was a kind gift from Amano Pharmaceutical Co. (Nagoya, Japan). Apolipoprotein (apo) CII was prepared from delipidated human very-low-density lipoproteins (Bengtsson and Olivecrona, 1980a).

Chemicals

Egg yolk phosphatidylcholine was from Sigma, St Louis, MO, USA; ~-a-dioleoyl-l-[~~C]glycerophosphocholine was from du Pont, NEN Research Products, MA, USA. Unlabeled trioleoylglycerol was bought from Nu Check Prep., Elysian, MN, USA ; [9,1 0-3H]oleic-acid-labeled trioleo ylglycerol was kindly prepared by Lennart Krabisch at the Department of Physiological Chemistry, University of Lund, Sweden. Bovine serum albumin (fraction 5) was from Sigma and heparin was from Lovens Likemedel, Malmo, Sweden.

Preparation of liposomes and emulsions

Liposomes were prepared according to a procedure published by Hamilton and Small (1981). Briefly, phosphatidylcholine (usually 50 mg) and trioleoylglycerol, in various amounts, were mixed while still in organic solvent and dried under nitrogen in a glass ampule. The dried lipid mixture was vacuum dried overnight and 4 m120 mM Tris/Cl, pH 7.4, 1% KCI 1 mM EDTA (buffer A) were added. The ampule was flame-sealed and the mixture was allowed to hydrate for at least 2 h. After hydration, another 4 ml buffer A was added and the mixture was sonicated for 5 min while the container was submerged in ice-cold water. Following sonication, the sample was centrifuged in a table-top centrifuge at low speed (2000 rpm, 10 min) to precipitate titanium particles coming from the sonicator probe during sonication. The sample was then ultracentrifuged for 4 h at 180000 xg. Fractions of the ultracentrifuged solution were separated by cutting the centrifuge tube in cross sections with a blade. In this system triacylglyerol-rich particles floated at the top whereas liposomes remained in the infranatant. We tested the stability of liposomes after isolation by ultracentrifugation and obtained the same kinetic results with freshly prepared liposomes and liposomes that had been stored at 4°C for one month.

Lipid analysis

Phosphatidylcholine was measured using an enzymatic kit from Wako Chemicals GmbH, FRG. In this assay, the phospholipid is hydrolyzed to free choline by phospholipase D. Choline is then oxidized to betaine by choline oxidase with the concomitant stoichiometric formation of hydrogen peroxide. The hydrogen peroxide is then used to generate a chromophore which is measured. When using liposomes, this assay gave the same results as those from the chemical procedure described by Bartlett (1959).

The concentration of trioleoylglycerol was determined from radioactivity measurements. A large stock solution of known amounts of unlabeled and 3H-labeled trioleoylglycerol was prepared and the radioactivity of several aliquots was measured to obtain an average radioactivity that would correspond to the known mass of trioleoylglycerol in the aliquot.

Kinetic studies

The medium for incubations with lipoprotein lipase or hepatic lipase contained 0.15 M Tris/Cl, pH 7.4, 15 IU/ml heparin, and 0.1 M NaC1. Concentrations of additional

components in the mixture and times of incubation are detailed in legends to the figures. All incubations were carried out at 25 "C. After incubation, 3H-labeled fatty acids released from trioleoylglycerol or phosphatidylcholine were extracted from 0.2 ml of the reaction mixture. We used the method described by Spooner et al. (1977) when studying only trioleoylglycerol hydrolysis and the method developed by Belfrage and Vaughan (1 969) when the experiment involved the determination of the rate of phosphatidylcholine hydrolysis alone or together with the rate of trioleoylglycerol hydrolysis (double-label experiments). 1 unit (U) lipase activity is 1 pmol fatty acid released/min at 25°C.

For experiments with lipoprotein lipase, an aliquot of the stock enzyme solution (0.3-0.7 mg/ml in 10 mM Bistris, pH 6.5, 1.0 M NaC1) was diluted in 5 mM deoxycholate, 0.1 mM SDS, 20 mM Tris, pH 8.5 (buffer B; Bengtsson and Olivecrona, 1979b). This dilution gives a stable enzyme solution from which suitable volumes (typically 5 kl) may be added without further dilution to the reaction mixture (final volume 0.2 ml). The final enzyme concentrations are specified in the figure legends. In control experiments with the various types of liposomes and triacylglycerol-rich particles, we added enzyme diluted in detergent-free buffer and studied the effects of addition of deoxycholate buffer. These experiments showed that even when 10 p1 buffer B was added (i.e. twice the volume used in our kinetic experiments), the activity of the enzyme was not affected.

To study if lipoprotein lipase was stable in the liposome system, we ran parallel incubations of the enzyme in medium with unlabeled phosphatidylcholine liposomes and in medium with unlabeled IntralipidR. It is known that under these conditions the enzyme is stable in the IntralipidK system (Bengtsson and Olivecrona, 1982). At the end of different incubation times (0 - 30 min), a [3H]trioleoylglycero1 lipid emulsion was added to each system (for the 0-min incubation, lipoprotein lipase was added last to a solution already containing the 3H-labeled emulsion). After this, the hydrolysis reaction was allowed to continue for another short incubation period (7 rnin). When 3H-labeled trioleoylglycerol hydrolysis was measured, lipoprotein lipase showed the same activity irrespective of the time and the system in which it was incubated.

Data analysis

curves to obtain apparent K, and V,,, values. We used Hanes plots (S/v vs S') from the data for substrate

RESULTS

Characterization of the liposome system

We prepared phosphatidylcholine liposomes with different amounts of trioleoylglycerol incorporated into the lipid bilayer as substrates for lipoprotein lipase. Mixtures with different proportions of trioleoylglycerol and phosphatidylcholine were dried under vacuum, hydrated with buffer, sonicated then ultracentrifuged. Previous work, where liposomes have been prepared and ultracentrifuged in the same manner, has shown that excess trioleoylglycerol partitions at the top. The bulk of the tube contains a solution of liposomes homo- geneous in size, and the bottom of the tube contains a phosphatidylcholine precipitate (Hamilton and Small, 1981).

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30 I t

Bottom

Section of Centrifuge Tube

Fig. 1. Separation of sonicated lipid mixtures by ultracentrfigation. Mixtures of trioleoylglycerol and phosphatidylcholine were dried, hydrated, sonicated in buffer, ultracentrifuged and the trioleoylglycerol content determined as specified in Experimental Procedures. The figure shows results obtained when the approximate amounts of trioleoylglycerol in the original mixture were 30Y0 ( O ) , 10% (0) and 1 YO (A). Upper fractions of the centrifuge tube with values greater than 3 mo1/100 mol trioleoylglycerol contain emulsion droplets and liposomcs (level indicated by arrow) whereas fractions with lower value contain only liposomes

Fig. 1 shows the amount of trioleoylglycerol in sections of the centrifuge tube going from top to bottom. When the starting lipid mixture contained 30% trioleoylglycerol, fraction 1, the top fraction of the centrifuge tube, contained an emulsion in which triacylglycerols made up 35% of the lipid molecules. This fraction also included a small amount of the bulk solution containing liposomes. Lower fractions showed less and less trioleoylglycerol until the amount leveled off at about 3 YO of total lipid. Recentrifugation of lower fractions containing amounts greater than 3% trioleoylglycerol resulted in additional partitioning of trioleoylglycerol to the top of the solution. On the other hand, recentrifugation of lower fractions where the amount of trioleoylglycerol was 3 mol/ 100 mol or less, did not result in further separation of trioleoylglycerol. Fig. 1 also shows that when the starting lipid mixture contained 10% trioleoylglycerol (0), there was also a top fraction with trioleoylglycerol in excess of 3%, but the rest of the tube contained fractions with a constant proportion of trioleoylglycerol/phosphatidylcholine, where triacylglycerol made up 3% or less of the lipid present. When using less than 3% trioleoylglycerol in the original lipid mixture (Fig. l), the lower fractions with a constant proportion of triacylglycerol/phospholipid reflected the amount of trioleoylglycerol initially added in the mixture. This allowed us to manipulate, to a certain extent, the amount of trioleoylglycerol that went into the lipid bilayer. Hamilton and Small (1981) have shown by NMR that the maximum solubility of trioleoylglycerol in the lipid bilayer of liposomes like those we report here, is about 2.8%. Our determinations of trioleoylglycerol present in the liposomes are consistent with this result.

The structures of emulsion droplets and liposomes are illustrated schematically in Fig. 2, which emphasizes the idea that the surface lipid composition of liposomes and emulsion droplets may be similar, whereas the inside of the macromolecular structures is different; emulsion droplets contain a hydrophobic core made up of trioleoylglycerol molecules whereas liposomes encapsulate aqueous buffer.

Hydrolysis of trioleoylglycerol incorporated in the liposome bilayer was linear with respect to lipoprotein lipase concentration and time of incubation under the conditions used (not shown). In most experiments less than 3% of the

ester bonds in the starting material were hydrolyzed. Other experiments indicated that linearity was maintained even at 15% triacylglycerol hydrolysis. The liposome system was sen- sitive to changes in pH and salt concentration; rates of hydrolysis were higher at higher pH in the range 6.8-8.0, and decreased at higher salt concentrations in the range 0.04- 0.29 M NaCl. We have chosen to run our experiments at pH 7.4 and 0.15 M NaCl to ensure that our mechanistic con- clusions can be seen within a physiological context. Other control experiments have shown that differences in the concentrations of Tris/Cl buffer and albumin do not have any effect on the rate when they are in the range 75 - 150 mM and 1 - 6% respectively.

Effect ojapoCII and albumin on lipoprotein lipase catalyzed hydrolysis

The hydrolysis reaction was strongly stimulated by addition of apoC1I. Fig. 3 illustrates this point; as the concentration of apoCII was increased, the reaction approached a maximal rate. The optimum apoCII concentrations for our liposome system were in the 4.4 - 20 pg/ml range; higher concentrations of apoCII were required to obtain maximum activation at higher liposome concentrations. This is in accord with the point made by Jackson et al. (1986) that the relevant measure of apoCII is its two-dimensional concentration at the lipid/water interface, not the bulk concentration. Based on the apoCII concentrations mentioned above, maximum activation with liposomes was reached when the molar ratio of apoCII/phosphatidylcholine was approximately 1 : 400.

In order to obtain optimal rates, apoCII and albumin were usually present in experiments with lipoprotein lipase. Fig. 4A shows the effect of the presence and absence of apoCII and albumin on trioleoylglycerol hydrolysis in liposomes. As ex- pected, the highest rates were obtained when apoCII and albumin were both present in the reaction mixture. When albumin was taken out of the system the rate decreased significantly, but trioleoylglycerol hydrolysis was still appreciable and progressed linearly. This is in contrast to previous results with IntralipidR emulsion droplets where it was found necessary to have albumin in the reaction mixture in order to observe apoCII activation (Bengtsson and Olivecrona, 1980 b). The lowest reaction rates were obtained in the absence of apoCII. Fig. 4B shows the corresponding results for phosphatidylcholine hydrolysis. Again, apoCII appeared to be more important than albumin for the progress of hydrolysis. In order to study if the decrease in rate in the absence of albumin was due to enzyme denaturation, the following experiment was carried out. In two parallel incubations lipoprotein lipase was incubated with liposomes with or without albumin. In the absence of albumin hydrolysis was very slow, but when albumin was added after 22 min the rate of hydrolysis immedi- ately started to increase and soon reached the same rate as when albumin was present from the start. Hence, the effect of albumin was not on enzyme stability. Albumin’s probable role is to remove fatty acid product from the bilayer.

Rates of triacylglycerol hydrolysis in triacylglycerol-rich particles and liposomes

We studied lipoprotein-lipase-catalyzed triacylglycerol hydrolysis in triacylglycerol-rich particles and liposomes. Sub- strate curves as illustrated in Fig. 5 could be fitted to the Michaelis-Menten equation. Apparent K, values, given in terms of phosphatidylcholine concentration, were consistently

318

It----------Onm-----------ll 25nrn-

Fig. 2. Schematic illustration of liposomes and emulsion droplets. The diameter of liposomes prepared in the same way as we do here has been reported by Huang (1969) to be about 25 nm. For emulsion droplets, we use 30 nm as the diameter for illustration purposes. The amount of trioleoylglycerol in the liposome bilayer has been drawn to correspond to the reported maximum solubility, about 3% (Hamilton and Small, 1981). The droplets have been drawn to contain the same proportion of trioleoylglycerol at the surface. However, trioleoylglycerol in the liposome bilayer accounts for all trioleoylglycerol available, whereas in the emulsion droplets the bulk of trioleoylglycerol is in the core

32t I

0.4 0.8 1.2 1.6

Apo CII (nmol/ml)

Fig. 3. Stimulation by apoCII of lipoprotein-lipase-catalyzed hydrolysis of trioleoylglycerol in liposomes. Liposomes (2.5 mo1/100 mol trioleoylglycerol, 3H-labeled and unlabeled) were incubated with different concentrations of apoCII in the presence of 60 mg albumin/ml. Final concentrations of phosphatidylcholine were 0.2 mM (0) and 0.7 mM (0) . Concentrations of other components are given in Exper- imental Procedures. The total volume was 0.2 ml. The reaction was started by adding lipoprotein lipase (LPL, 4.2 ng/ml) to the reaction mixtures. Data points arc thc averages of three separate determinations

similar for liposomes and triacylglycerol-rich particles in many experiments of this kind and varied in the range 0.06- 0.1 mM. The apparent V,,, was 10-fold higher for triacylglycerol-rich particles than for liposomes.

Fig. 6 shows hydrolysis of the phosphatidylcholine and trioleoylglycerol (1.1 mo1/100 mol) moieties in mixed liposomes by lipoprotein lipase (Fig. 6 A) and hepatic lipase (Fig. 6B). For both lipases, the amount of liposomes needed to reach substrate saturation was similar for triacylglycerol hydrolysis as for phosphatidylcholine hydrolysis, but this amount was higher for hepatic lipase than for lipoprotein lipase. For lipoprotein lipase the maximal rate for phosphatidylcholine hydrolysis was about eightfold higher than that for trioleoylglycerol hydrolysis. For hepatic lipase, the ratio between the rates for phosphatidylcholine hydrolysis/ trioleoylglycerol hydrolysis was 20 - 25-fold for all liposome concentrations. Since we used the same liposome preparation to study the two enzymes, it is clear that the ratio between the rates of phosphatidylcholine hydrolysis/trioleoylglycerol

hydrolysis was significantly higher for hepatic lipase than for lipoprotein lipase.

In contrast to the results with liposomes, both enzymes hydrolyzed triacylglycerol faster than phosphatidylcholine in a centrifuged fraction of triacylglycerol-rich particles containing 47 mo1/100 rnol trioleoylglycerol. For lipoprotein lipase, trioleoylglycerol hydrolysis reached a value of 75 U/mg enzyme and there was no detectable phosphatidylcholine hydrolysis. The lowest detectable activity for phosphatidylcholine hydrolysis under these conditions would be around 7 U/mg enzyme; this means that phosphatidylcholine hydrolysis must have been less than 10% of the value obtained for trioleoylglycerol. For hepatic lipase, the ratio between the rates of trioleoylglycerol hydrolysis/phosphatidylcholine hydrolysis in the triacylglycerol-rich fraction was similar (3.8 _+ 0.7) at all substrate concentrations studied. Since this ratio was about 0.05 for liposomes containing 1.1 mol/ 100 mol trioleoylglycerol (see above), trioleoylglycerol hydrolysis increased more than 60-fold with respect to phosphatidylcholine hydrolysis in going from the liposomes to the triacylglycerol-rich particles. For hepatic lipase, substrate saturation was not reached with the triacylglycerol-rich particles. At the two highest concentrations used the rates of trioleoylglycerol hydrolysis reached 290 mU/ml and 366 m u / ml enzyme. These values are about 20-fold higher than the rates of trioleoylglycerol hydrolysis at comparable concentrations of liposomes.

We also used mixed liposomes and the triacylglycerol-rich particles as substrates for bacterial lipase from P.fluorescens. With this enzyme, there was no detectable phospholipid hydrolysis in liposomes or triacylglycerol-rich particles. The rate of trioleoylglycerol hydrolysis was about 100 times higher (40 U/mg enzyme) with the triacylglycerol-rich fraction than with the liposomes, but with neither type of substrate did we reach substrate saturation of the enzyme.

The rate of trioleoylglycerol hydrolysis catalyzed by lipoprotein lipase increased with increasing amounts of trioleoylglycerol incorporated into the liposome bilayer (Fig. 7). Similar observations have been made for monolyer systems by Demel et al. (1984, 1985). As mentioned above, the rate of phosphatidylcholine hydrolysis was about eight times the rate of trioleoylglycerol (Fig. 6A) when there was 1.1 mo1/100 mol trioleoylglycerol in the bilayer. Ifwe consider that there is a %-fold difference in surface concentration between the two lipids and only an eightfold difference in rate,

319

240

200-

160-

240[ V,,, TG-Rich Particles

A -

l;:L 40

5 10 15 20 25

Time (min) !z

,zooor 1600 /

2. 10 20 30 40 50 60

Y Time (min) .-

Fig. 4. &Pci of apoCII and albumin on hydrolysis in liposomes. (A) Trioleoylgl ycerol hydrolysis. [ 3H]Trioleoylglycerol-labeled liposomes containing 1.4 mo1/100 mol trioleoylglycerol were used. The final concentration of liposomes was 0.7 mM phosphatidylcholine. When prc- sent, the concentrations of albumin and apoCII were 60 mg/ml and 8.8 pg/ml, respectivcly. The reaction was started by adding lipoprotein lipase (0.2 ng/ml for incubations with apoCII or 3.4 ng/ml for incubations without apoC1I). Final volume was 2 ml; 0.2-ml aliquots were withdrawn from the reaction mixture at the times indicated. ( 0 ) apoCI1 and albumin; (0) apoCI1 but no albumin; ( A ) albumin but no apoCII; (0) no apoCII or albumin. (B) Phosphatidylcholine hydrolysis. [ ‘‘C]Phosphatidylcholine-labeled liposomes, with approximately 2.5 mo1/100 mol unlabeled trioleoylglycerol incorporated in the lipid bilayer, wcre used in this experiment. Final concentrations of phosphatidylcholinc were 0.4 mM for incubations with apoCIl and 1.2 mM for incubations without apoC11. When present, conccntrations of albumin and apoCII were 30 mg/ml and 4.4 pg/ml, respectively. The reaction was started by adding lipoprotein lipase (0.7 ng/ml for incubations with apoCl1 or 14 ng/ml for incubations without apoCII). Other conditions and labels as in (A)

it is clear that even in the case of liposomes, trioleoylglycerol is the preferred substrate for lipoprotein lipase.

DISCUSSION

In this study we have used liposomes containing trace amounts of triacylglycerol integrated in the bilayer as substrates for lipoprotein lipase. The liposomes proved t o be a good and stable substrate for the lipase, and gave new information about some major questions in lipase research.

Lipoprotein-lipase-catalyzed hydrolysis of liposomes progressed well and was greatly stimulated by apoCII even without albumin. This is in marked contrast t o findings with lipoproteins and emulsions where fatty acids strongly inhibit the progress of hydrolysis (Scow and Olivecrona, 1977; Bengtsson

80

1 6 [ (

0.2 0.4 0.6 [Phosphatidylcholine](mM)

Fig. 5. Kinetics with liposornes and triacylglycerol-richparticles. Differ- ent concentrations of a triacylglycerol (TG)-rich fraction ( 0 ) or liposomes (0) were incubated with lipoprotein lipase (3.4 ngiml). The triacylglycerol-rich fraction is the top fraction (fraction 1) of a centrifuge tube obtained after ultracentrifugation of a sonicated mixture of phosphatidylcholinc and trioleoylglycerol (unlabeled and 3H labeled) in aqueous buffer. All conditions were similar as those used to prepare liposomes (Experimental Procedures) except that sucrose ( 1 00 mg/ml) was added to the sonicated mixture which was then layered under an equal volume (2 ml) of aqueous buffer. Sucrose gradients have becn used successfully to isolate triacylglycerol-rich particles with low amounts of phosphatidylcholine (Bengtsson and Olivecrona, 1980a). Lipid analysis of fraction 1 showed it contained 70% triacylglycerol. Liposomes contained 2.6 mol/lOO mol trioleoylglycerol in the lipid bilayer. Final volume of reaction mixture was 0.2 ml. Reaction mixture contained albumin (60 mg/ml) and apoCII (8.8 pg/ml) in addition to the usual componcnts. Incubation times were 7 min and 25 min for triacylglycerol-rich particles and liposomcs, respectively. Each data point is the average of three separate assays. Thc concentration of lipid substrate is given in terms of phosphatidylcholine concentration. because this is the concentration that reflects the surface area of the macromolecular substrate. Calculated apparent Vmax values are indicated in the figure and were 20 U/mg and 220 U/mg lipoprotein lipase for liposomes and triacylglycerol-rich particles, respectively

and Olivecrona, 1980b), and impede the stimulation by apoCII (Bengtsson and Olivecrona, 1979a). The present results support the view that the fatty acid inhibition is mainly a physical effect caused by conversion of core triacylglycerols t o fatty acids and monoglycerides which form extensions of the surface film, and separate bilayered structures (Scow and Blanchette-Mackie, 1985; Cistola e t al., 1988), which sequester the lipase. This is not seen with liposomes where the substrate molecules are surface-located, and hydrolysis does not cause overcrowding of the surface.

The two major findings here were that triacylglycerol hydrolysis proceeded much faster in emulsion droplets than in the mixed liposomes, and that whereas triacylglycerol hydrolysis was favored over phosphatidylcholine hydrolysis in emulsion droplets, the reverse was true in liposomes. Studies with monolayers have shown that lipoprotein lipase action is dependent on surface packing and composition (Jackson et al., 1980; Vainio et al., 1983a, 1983b; Demel e t al., 1984; Demel and Jackson, 1985). Can such differences between the quality of the interface explain the large differences in hydrolysis observed between the mixed liposomes and emulsion droplets? Small, Hamilton and their coworkers (Hamilton, 1989; Hamilton and Small, 1981,1982; Hamilton et al., 1983; Miller and Small, 1987) have provided physicochemical consider- ations and da ta which argue that the surfaces of the two types of particles are similar. We consistently found that substrate curves were similar for droplets and liposomes, when the concentrations were expressed in terms of phospholipid (which should reflect the surface area). This indicates that the

3 20

0.4 0.8 1.2 1.6

[Phosphatidylcholine] (mM)

Fig. 6. Pliosplzutidyl~koline and trioleoylglycerol hydrolysis in lipo- .somes. [3H]Trioleoylglycero1 (1 , I mo1/100 mol) and ['4C]phosphatidylcholine-labeled liposomes were used in these experiments to allow determination of the rate of hydrolysis of phosphatidylcholine ( 0 ) and trioleoylglycerol(0) in the same liposomes. (A) Incubations with lipoprotein lipase (LPL). 1.7 ng enzyme/ml was used for trioleoylglycerol hydrolysis. A higher enzyme conccntration, 6.8 ng/ ml was used for studies of phosphatidylcholine hydrolysis because this substrate had much lower specific radioactivity. The reaction mixture contained, in addition to the usual components listed in Experimental Procedures, albumin (30 mg/ml) and apoCII(8.8 pg/ml at all liposome concentrations except at 1.8 mM phosphatidylcholine where 20 pg/ml was used). Final volume was 0.2 ml; incubation time was 60 min for phosphatidylcholine hydrolysis and 40 min for trioleoylglycerol hydrolysis. Data points are the average of three separate determinations. (€3) Incubations with hepatic lipase (HL). A solution of hcpatic lipase was used in sufficient amount ( 5 PI) to give the ratcs shown. Hydrolysis rates of both lipids wcrc dctermincd concomitantly this timc. Incubation time was 40 min; all other conditions were the same as in (A), except that there was no apoCI1 in thc rcaction mixturc

lipase experienced the surfaces of the two types of particles as similar with respect to binding. We can not, however, exclude the possibility that there are subtle differences between the surfaces, that are not evident in physical studies, and are not reflected in lipase binding, but radically influence catalysis.

The recent resolution by X-ray crystallography of the folded structure for pancreatic lipase has shown that the active site is at the bottom of a narrow cleft (Winkler et al., 1990). The precise arrangement with respect to the lipid/water interface is not yet clear. With liposomes (and monolayers), substrate molecules would have to slide into the active site from the side, or the enzyme would have to jump randomly from hydrolyzed products to new surface-located substrate molecules. Any of these models cold explain why the lipase hydrolyzed mainly phospholipids when it acted on the liposomes. Our data suggest, however, that entry of substrate molecules from below is the favored pathway. With emulsion droplets this pathway could provide a kinetic advantage for triacylglycerol compared to phospholipid entry into the active site, explaining

0

3

I

1 2 3

Trioleoylglycerol (mo1/100 mol)

Fig. 7. Rate of trioleoylglycerol hydroij~sis for dilyerent amounts of trioleoylglycerol in liposome hilayer. Each point corresponds to an apparent V,,, value obtained from substrate curves as in Fig. 5. All points correspond to determinations made with [3H]trioleoylglycero1- labeled liposomes except the rate corresponding to 1.1 YO trioleoylglycerol where we included the result for trioleoylglycerol hydrolysis from Fig. 6 A where doubly labeled liposomes were used

why the ratio of triacylglycerol/phospholipid hydrolysis was much higher with the droplets than with liposomes. In this connection it is interesting to note a mathematical study of the kinetics of triacylglycerol lipolysis in rat chylomicra by Foster and Berman (1981). They found it necessary to include a direct pathway from triacylglycerol to monoacylglycerol in their model. The physical equivalent of this is that the diacylglycerol formed by one hydrolytic cycle does not mix back with other diacylglycerols in the particles, but is preferen- tially hydrolyzed. This supports the hypothesis that the path- ways of substrate entry and product exit from the active site are restricted. An implication is that the enzyme remains at the interface while carrying out several, perhaps many, hydrolytic events.

The data revealed differences between the lipases in substrate selection. The bacterial lipase hydrolyzed only triacylglycerols, and showed no activity at all against phospholipids. Lipoprotein lipase and hepatic lipase, which are closely related, hydrolyzed both lipids, but the hepatic lipase consistently showed a higher ratio of phospholipid/triacylglycerol hydrolysis. This must reflect some subtle difference between the two lipases. The liposome system should be well suited for detailed comparison of substrate preference between lipases, and to compare hydrolysis of different molecular species, e. g. triacylglycerols with different fatty acid patterns, in a system where the surface concentration of each could be known.

The large difference in lipase action on mixed liposomes and emulsion droplets could not have been anticipated from current theories which emphasize that lipolysis is an interface reaction (Verger and deHaas, 1976; Brockman, 1984), and that the surface composition and properties are similar for phospholipid-enveloped triacylglycerol droplets as for phospholipid liposomes containing saturating amounts of triacylglycerol (Hamilton and Small, 1981 ; Miller and Small, 1987). Our finding suggest that for lipoprotein lipase it is not only the quality of the interface that matters, but also the chemical composition below. Similar results were obtained with the related hepatic lipase, and with an unrelated bacterial lipase. Thus, the observations may be relevant for the mode of action of many lipases.

This work was supported by grants from the Swedish Medical Research Council (B13X-727) and from the Swedish Margarine In- dustry Fund for Research in Nutrition.

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Comparison of the action of lipoprotein lipase on triacylglycerols and phospholipids when presented...

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