Binding to heparan sulfate is a major event during catabolism of lipoprotein lipase by HepG2 and other cell cultures.

Lipoprotein lipase (LPL) is rapidly and efficiently cleared from the circulation by the liver through an as yet unclear mechanism. In the present study, we determined the nature of LPL interactions with the liver parenchimal cell line HepG2 as compared to other cells in culture. Binding, cell association and degradation of 125I-labelled bovine milk LPL by HepG2 cells, normal and low density lipoprotein (LDL) receptor-negative human fibroblasts and Chinese hamster ovary (CHO) cells show similar values irrespective of source and origin. LPL metabolism in HepG2 cells was characterized by a high capacity to degrade the enzyme, an extremely high sensitivity to heparin and was inhibited by 60%-70% after treatment of the cells with sodium chlorate and heparinase (but not chondroitinase). These findings suggested an important role for heparan sulfate in the process of cell interaction and metabolism of LPL. To further clarify the role of heparan sulfate in determining the LPL-cell interactions, we compared the metabolism of LPL in wild type and mutant heparan sulfate-deficient CHO cells. Heparan sulfate-deficient CHO cells show a low capacity to bind and degrade LPL, about 10%-20% that of the wild type cells. In another set of experiments, we sought to determine whether LPL interactions with HepG2 cells are affected by triglyceride-rich lipoproteins. The results clearly show that whereas unlabeled LPL dramatically enhanced the metabolism of radioiodinated very low density lipoprotein (VLDL), unlabeled VLDL had no effect on radioiodinated LPL metabolism in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of lipoprotein lipase to alpha 2-macroglobulin.

The interaction between bovine lipoprotein lipase (bLPL) and human alpha 2-macroglobulin (alpha 2M) was studied by use of non-denaturing PAGE and gel-permeation, Zn(2+)-Sepharose and heparin-Sepharose chromatography. It was demonstrated that bLPL in vitro binds non-covalently to native alpha 2M, but not to the receptor-recognized form produced by treatment of alpha 2M with chymotrypsin or methylamine. A small amount of bLPL was bound covalently to alpha 2M by disulphide interchange, when incubated together with chymotrypsin or methylamine. Whereas alpha 2M in complex with bLPL still bound to Zn(2+)-Sepharose, bLPL lost the ability to bind to heparin-Sepharose. Preincubation of bLPL with heparin prevented complex-formation with alpha 2M, suggesting that alpha 2M interacts with the heparin-binding domain of bLPL. Experiments in which 125I-bLPL was incubated with human plasma at 20 degrees C demonstrated an 11-17% binding of the labelled lipase to alpha 2M, indicating that this interaction may be of physiological significance.

Lipoprotein lipase in human plasma is mainly inactive and associated with cholesterol-rich lipoproteins.

This study was designed to further ascertain the presence in plasma of lipoprotein lipase (LPL) bound to circulating lipoproteins. Lipoprotein lipase mass and activity values in preheparin plasma from 20 volunteers were 69.8 +/- 6.6 ng.ml-1 and 1.54 +/- 0.15 mU.ml-1, respectively, and no significant correlation between mass and activity was observed. Fifteen min after heparin injection, LPL mass had increased to 536 +/- 60 ng.ml-1 and LPL activity to 261 +/- 34 mU.ml-1 and a highly significant correlation between the increments in mass and activity was observed. The released material had a specific activity of 0.57 +/- 0.03 mU.ng-1. The LPL mass in preheparin plasma eluted early from heparin-Sepharose, in the position expected for inactive LPL monomers. Western blot analysis showed that the eluted material had the size expected for the LPL subunit (55 kDa). The increment of mass and activity after heparin eluted later from heparin-Sepharose, in the position expected for active LPL dimers. It is concluded that preheparin plasma contains substantial amounts of inactive LPL protein, and that heparin releases mainly active LPL into circulation. On gel filtration LPL activity and mass in postheparin plasma eluted mainly in the positions of LDL and HDL. Electron microscopy of immunostained fractions showed reaction for LPL and apolipoprotein B, or apolipoprotein A-I, on the same particles. LPL mass in preheparin plasma eluted in a similar pattern, associated with LDL and HDL. In postprandial plasma substantial amounts of LPL protein eluted with the triglyceride-rich lipoproteins. When 125I-labeled bovine LPL was added to plasma or to ultracentrifugally isolated lipoproteins and then analyzed by gradient gel electrophoresis, the labeled lipase moved with the lipoproteins. The presence of substantial amounts of inactive LPL protein associated with lipoproteins in plasma may have important implications for the metabolism of the particles in view of recent reports on avid binding of LPL-lipoprotein complexes to cell surfaces and receptors.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds lipoprotein lipase and beta-migrating very low density lipoprotein associated with the lipase.

Lipoprotein lipase (LPL) causes a marked increase in the cellular binding of beta-migrating very low density lipoprotein (beta-VLDL) to a large receptor compatible with the alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) (Beisiegel, U., Weber, W., and Bengtsson-Olivecrona, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8342-8346). Here we demonstrate that LPL binds to the alpha-chain of purified alpha 2MR/LRP immobilized on microtiter plates. The binding, apparently to multiple sites, was blocked by heparin and inhibited by the alpha 2MR-associated protein (alpha 2MRAP) and by EDTA. Immobilized LPL bound alpha 2MR/LRP in solution as well as beta-VLDL prepared from cholesterol-fed rabbits. Both binding reactions were dependent on an intact carboxyl-terminal folding domain of LPL, but were independent of its dimeric structure and intact catalytical function. Dimeric LPL could mediate binding of beta-VLDL to immobilized alpha 2MR/LRP and to cells, e.g. monocytes. In contrast, LPL monomers were not able to mediate binding to immobilized alpha 2MR/LRP, presumably because of cross-inhibition due to close relation between the binding regions for the lipoprotein and for the receptor in the carboxyl-terminal domain of the LPL monomer. Heparin, but not alpha 2MRAP, inhibited cellular binding of 125I-LPL or 125I-beta-VLDL supplemented with LPL. However, alpha 2MRAP inhibited degradation of the two ligands by about 90% and 40-50%, respectively. The results show that LPL is a ligand for alpha 2MR/LRP and, because of its affinity for lipoprotein particles, dimeric LPL can mediate or strengthen binding of beta-VLDL to this receptor. It is proposed that LPL binds primarily to cell surface heparan sulfate in monocytes and is presented for endocytosis and degradation by alpha 2MR/LRP. Moreover, beta-VLDL may be further supplemented with LPL at the cell surface and achieve affinity for alpha 2MR/LRP.

Changes in biological activity and immunoreactive mass of lipoprotein lipase in congenital nephrosis: relationship to hypertriglyceridaemia.

The major lipid disturbance in children with congenital nephrosis of the Finnish type (CNF) is hypertriglyceridaemia. To determine whether or not hypertriglyceridaemia is caused by defective triglyceride catabolism, we measured lipoprotein lipase (LPL) activities and masses at various stages of the disease. At age 3 months in CNF both LPL activity and mass were decreased, but a close positive correlation between these parameters similar to that in controls was observed. At age 9 months both LPL activity and mass were even lower. At that time a significant positive correlation (r = 0.72, P < 0.05) between LPL activities and albumin concentrations and significant negative correlations between plasma free fatty acid (FFA) concentrations and LPL activities (r = -0.72, P < 0.05) and between plasma FFA concentrations and serum albumin concentrations (r = -0.73, P < 0.05) were observed, suggesting that low albumin concentrations result in increase of FFA levels, which could interfere with a normal LPL function at the endothelial surface. On dialysis after nephrectomy, LPL activities and masses increased. At age 3 and 9 months apoprotein C-II (apo C-II) and apoprotein C-III (apo C-III) levels were not decreased although apoproteins were being lost into the urine. On dialysis the mean ratio of apo C-II/C-III was significantly lower than the mean in controls (P < 0.001). We conclude that impaired function of LPL seems to be the major cause of hypertriglyceridaemia and disintegrity of the VLDL-IDL-LDL delipidation cascade in children with CNF.

Assembly of lipoprotein lipase in perfused guinea-pig hearts.

It has been suggested that lipoprotein lipase (LPL) can be assembled into its catalytically active dimeric form only after its oligosaccharide chains have been processed in the Golgi. To study this in a complete organ, LPL was metabolically labelled with [35S]methionine in perfused guinea-pigs hearts. After 10 min pulse-labelling, LPL protein was eluted as two peaks from heparin-agarose: peak 1 at about 0.65 M NaCl, peak 2 at about 0.95 M NaCl. Catalytic activity was associated only with peak 2. Model studies with bovine LPL showed that active dimeric LPL is eluted in peak 2, but after treatments that dissociate the enzyme into inactive monomers it is eluted in peak 1. Pulse-labelled LPL in both peak 1 and peak 2 was fully sensitive to treatment with endoglycosidase (Endo) H. With chase, peak 1 disappeared and peak 2 acquired resistance to Endo H. These findings suggest that core glycosylated LPL is assembled into dimers already in the endoplasmic reticulum and that processing of the oligosaccharide chains occurs after dimerization.

Chymotryptic cleavage of lipoprotein lipase. Identification of cleavage sites and functional studies of the truncated molecule.

Treatment of bovine lipoprotein lipase (LPL) with chymotrypsin results in cleavage between residues Phe390-Ser391 and between Trp392-Ser393, indicating that this region is exposed in the native conformation of LPL. Two main fragments are generated, one large including the amino-terminus (chymotrypsin-truncated LPL = c-LPL) and one small, carboxy-terminal fragment. The small fragment is not stable, but is further degraded by the protease. Isolated c-LPL has full catalytic activity against tributyryl glycerol (tributyrin) and p-nitrophenyl butyrate, while the activity against emulsions of long-chain triacylglycerols and against liposomes is reduced and the activity against milk fat globules and chylomicrons is lost. Several properties of c-LPL were investigated. It was found that c-LPL interacts with apolipoprotein CII (apo CII) as efficiently as intact LPL. The truncated enzyme bound to liposomes and to emulsions of long-chain triacylglycerols as well as the intact enzyme did. In contrast, c-LPL did not bind to milk fat globules or to chylomicrons. The activity of c-LPL was more sensitive to inhibition by other lipid-binding proteins, e.g. apolipoprotein CIII (apo CIII), than was the intact enzyme. The affinity for heparin was as high with c-LPL as with intact LPL. Like intact LPL, c-LPL is dimeric in its active form, as evidenced by sucrose density gradient centrifugation. It is concluded that the reduced catalytic and lipid-binding properties of c-LPL compared with intact LPL are related to the properties of the substrate interface. It is speculated that the carboxy-terminal part of LPL contains a secondary lipid-binding site, which is important for activity against chylomicrons and related substrates.

New aspects on heparin and lipoprotein metabolism.

Lipoprotein lipase (LPL) and hepatic lipase (HL) are two enzymes which participate in metabolism of plasma lipoproteins. The enzymes are located at vascular surfaces and are released from their binding sites on injection of heparin. In this paper we give a short overview of the structure of the lipases and their role in lipoprotein metabolism. Earlier studies had shown that low molecular weight (LMW) heparin preparations result in lower LPL activities in blood than do corresponding amounts of conventional heparin. Studies with organ perfusion in rats show that the two types of heparin have similar ability to release the lipases from their binding sites in extrahepatic tissues, but that LMW heparin is less effective than conventional heparin in preventing rapid uptake and degradation of LPL by the liver. After injection of heparin the metabolism of triglyceride-rich lipoproteins is initially accelerated, presumably as a result of the high levels of circulating LPL. Then follows a phase when lipoprotein metabolism is slower than normal, perhaps because endothelial LPL has been depleted by accelerated transport to and degradation in the liver.

Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins: role of lipoprotein and hepatic lipase.

To explore the interactions of triacylglycerol and phospholipid hydrolysis in lipoprotein conversions and remodeling, we compared the activities of lipoprotein and hepatic lipases on human VLDL, IDL, LDL, and HDL2. Triacylglycerol and phospholipid hydrolysis by each enzyme were measured concomitantly in each lipoprotein class by measuring hydrolysis of [14C]triolein and [3H]dipalmitoylphosphatidylcholine incorporated into each lipoprotein by lipid transfer processes. Hepatic lipase was 2-3 times more efficient than lipoprotein lipase at hydrolyzing phospholipid both in absolute terms and in relation to triacylglycerol hydrolysis in all lipoproteins. The relationship between phospholipid hydrolysis and triacylglycerol hydrolysis was generally linear until half of particle triacylglycerol was hydrolyzed. For either enzyme acting on a single lipoprotein fraction, the degree of phosphohydrolysis closely correlated with triacylglycerol hydrolysis and was largely independent of the kinetics of hydrolysis, suggesting that triacylglycerol removed from a lipoprotein core is an important determinant of phospholipid removal via hydrolysis by the lipase. Phospholipid hydrolysis relative to triacylglycerol hydrolysis was most efficient in VLDL followed in descending order by IDL, HDL, and LDL. Even with hepatic lipase, phospholipid hydrolysis could not deplete VLDL and IDL of sufficient phospholipid molecules to account for the loss of surface phospholipid that accompanies triacylglycerol hydrolysis and decreasing core volume as LDL is formed (or for conversion of HDL2 to HDL3). Thus, shedding of whole phospholipid molecules, presumably in liposomal-like particles, must be a major mechanism for losing excess surface lipid as large lipoprotein particles are converted to smaller particles. Also, this shedding phenomenon, like phospholipid hydrolysis, is closely related to the hydrolysis of lipoprotein triacylglycerol.

Sequence specific 1H-NMR assignments and secondary structure of a carboxy-terminal functional fragment of apolipoprotein CII.

The structural properties of a synthetic fragment of human apolipoprotein CII (apoCII) has been studied by circular dichroism and proton nuclear magnetic resonance. The fragment corresponds to the carboxy-terminal 30 amino acid residues and retains the ability of apoCII to activate lipoprotein lipase. Like native apoCII, the fragment has a tendency to self-associate in pure aqueous solution. Addition of 1,1,1,3,3,3-hexafluoro-2-isopropanol to aqueous solvent dissolves the aggregates and leads to an increase in the alpha-helical content of the peptide, probably by stabilizing transient helical structures. The resonances in the 1H-NMR spectrum of the fragment in 35% (CF3)2CHOH were assigned through standard procedures from nuclear Overhauser enhancement spectroscopy, correlated spectroscopy and total correlated spectroscopy experiments. The NMR data indicates the formation of a stable alpha helix spanning Ile66-Gly77. Another alpha helical turn may be formed between Lys55 and Ala59 and possibly span even further towards the carboxyl terminus. These structural elements are different from those previously predicted for this part of the sequence of apoCII.

Release of lipoprotein lipase to plasma by triacylglycerol emulsions. Comparison to the effect of heparin.

It was previously known that lipoprotein lipase (LPL) activity in plasma rises after infusion of a fat emulsion. To explore the mechanism we have compared the release of LPL by emulsion to that by heparin. After bolus injections of a fat emulsion (Intralipid) to rats, plasma LPL activity gradually rose 5-fold to a maximum at 6-8 min. During the same time the concentration of injected triacylglycerols (TG) decreased by about half. Hence, the time-course for plasma LPL activity was quite different from that for plasma TG. The disappearance of injected 125I-labelled bovine LPL from circulation was retarded by emulsion. This effect was more marked 30 min than 3 min after injection of the emulsion. The data indicate that the release of LPL into plasma is not solely due to binding of the lipase to the emulsion particles as such, but involves metabolism of the particles. Emulsion increased the fraction of labelled LPL found in adipose tissue, heart and the red muscle studied, but had no significant effect on the fraction found in liver. The effects of emulsion were quite different from those of heparin, which caused an immediate release of the lipase to plasma, decreased uptake of LPL in most extrahepatic tissues by 60-95%, and increased the fraction taken up in the liver.

Lipoprotein lipase in lungs, spleen, and liver: synthesis and distribution.

Lipoprotein lipase (LPL, E C 3.1.1.34) is the enzyme responsible for hydrolysis of triacylglycerols in plasma lipoproteins, making the fatty acids available for use by subjacent tissues. LPL is functional at the surface of endothelial cells, but it is not clear which cells synthesize the enzyme and what its distribution within tissues and vessels is. In previous studies we reported that in the major LPL-producing tissues (muscles, adipose tissue, and mammary gland) the enzyme is made by the major cell types. In the present work we have studied in adult guinea pigs some tissues that present LPL activity but in lower amounts (lung, spleen, and liver). On cryosections of these tissues we have searched for specific cell expression of the LPL gene (by in situ hybridization using a RNA probe) and for the corresponding protein distribution (by immunocytochemistry). Based on morphological criteria we can suggest that, contrary to the main LPL-producing tissues, in these tissues the enzyme is made by scattered cells, such as macrophages in the lung and spleen and Kupffer cells in the liver; endothelial cells present but do not synthesize the enzyme, indicating that the endothelial LPL originates in other cells. In the liver strong immunoreaction was detected in the sinusoid in contrast to the low level of mRNA expression, suggesting that liver takes up circulating LPL from blood.

Domain structure of endothelial heparan sulphate.

The domain structure of heparan sulphate chains from an endothelial low-density proteoglycan was examined using specific degradations of the chains while attached to the intact proteoglycan. 'Inner' chain fragments, remaining on the protein core, were separated from 'outer' fragments by gel chromatography, and were subsequently released from the protein core by alkaline cleavage. The structure of 'inner' and 'outer' chain fragments was then examined and compared. Using deaminative cleavage we obtained evidence that the first N-sulphated glucosamine residue is variably positioned some 10-17 disaccharides from the xylose-serine linkage of the proteoglycan. Digestion with heparinase yielded 'inner' and 'outer' fragments covering a broad range of different sizes, indicating a scarce and variable distribution of sulphated iduronic acid in the native chains. N-sulphated glucosamine occurred more frequently in the 'outer' fragments. We also studied the affinity of the endothelial heparan sulphate chains towards two presumptive biological ligands, namely antithrombin III and lipoprotein lipase. A major part of the endothelial heparan sulphate chains showed a weak affinity for antithrombin III and the affinity was essentially lost on heparinase digestion. On lipoprotein lipase-agarose the endothelial heparan sulphate chains were eluted at the same salt concentration as heparin, and the binding persisted, although with decreased strength, after digestion with heparinase.

Rat apolipoprotein C-II lacks the conserved site for proteolytic cleavage of the pro-form.

Apolipoprotein C-II (apoC-II) plays a critical role in the metabolism of plasma lipoproteins as an activator for lipoprotein lipase. Human apoC-II consists of 79 amino acid residues (pro-apoC-II). A minor fraction is converted to a mature form by cleavage at the site QQDE releasing the 6 amino-terminal residues. We have cloned and sequenced the cDNA for rat apoC-II from a liver cDNA library using human apoC-II cDNA as a probe. The cDNA encodes a protein of 97 amino acid residues including a signal peptide of 22 amino acid residues. There is approximately 60% similarity between the deduced amino acid sequence of rat apoC-II and other apoC-II sequences presently known (human, monkey, dog, cow, and guinea pig). Compared to these, rat apoC-II is one residue shorter at the carboxyl terminus. Furthermore, there is a deletion of 3 amino acid residues (PQQ) in the highly conserved cleavage site where processing from pro- to mature apoC-II occurs in other species. Accordingly, rat apoC-II isolated from plasma was mainly in the pro-form. Northern blot analyses indicated that rat apoC-II is expressed both in liver and in small intestine.

Lipoprotein lipase enables triacylglycerol hydrolysis by perfused newborn rat liver.

Fasted 1-day-old rat liver has high heparin-releasable (endothelial) lipoprotein lipase (LPL) activity, and its hepatocytes synthesize LPL protein. To test the physiological role of this LPL, we perfused the isolated organ with a 0.8 mM triacylglycerol (TAG) (Intralipid + glycerol tri[3H]oleate) 6.3% serum medium. Samples of the recirculated perfusate were taken at different times to determine 3H in TAG, free fatty acid (FFA), and water-soluble (WS) fractions. In the medium [3H]TAG disappeared and [3H]FFA and [3H]WS fractions appeared linearly with time. This TAG hydrolysis was 1) absent when medium was recirculated without liver, 2) not affected by chloroquine addition, 3) inhibited by anti-LPL immunoglobulins, 4) absent when serum was omitted from the medium, and 5) restituted when apolipoprotein CII was added to the medium without serum. Therefore, lysosomal lipase is not involved in this TAG hydrolysis, the features of which are characteristic of LPL, not of the so-called "hepatic endothelial lipase." Thus LPL activity enables the neonatal rat liver to hydrolyze and take up circulating TAG, i.e., has the same function as extrahepatic LPL.

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase.

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.

Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein.

Chylomicron catabolism is known to be initiated by the enzyme lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34). Chylomicron remnants, produced by lipolysis, are rapidly taken up by the liver via an apolipoprotein E (apoE)-mediated, receptor-dependent process. The low density lipoprotein (LDL) receptor-related protein (LRP) has been suggested as the potential apoE receptor. We have analyzed the binding of human chylomicrons to HepG2 cells in the absence and presence of lipoprotein lipase. Bovine and human lipoprotein lipases were able to increase the specific binding of the chylomicrons by up to 30-fold. This effect was not dependent on lipolysis but appeared to be due to the lipase protein itself. It was not found when a structurally unrelated, bacterial lipase was used. Using beta-migrating very low density lipoproteins (beta-VLDLs), known as a good ligand for LRP, binding studies were performed on LDL receptor-negative human fibroblasts. The binding was increased 40-fold by addition of lipoprotein lipase. Crosslinking experiments on cells with 125I-labeled apoE liposomes or lipoprotein lipase showed that both proteins were able to bind to LRP on the cell surface. The binding of apoE to LRP was highly increased by the addition of lipase. We conclude that lipoprotein lipase strongly enhances the binding of apoE-containing lipoproteins to LRP and therefore might play an important role in chylomicron catabolism not only because of its lipolytic activity but also because of its structural properties.

Lipoprotein lipases and vitellogenins in relation to the known three-dimensional structure of pancreatic lipase.

A 106-residue region of high similarity between lipoprotein/pancreatic/hepatic lipases and Drosophila vitellogenins encompasses four beta-strands with all residues but one strictly conserved or conservatively replaced between the structures, and enclosing the putative active site Ser-152. The properties suggest a common folding pattern but the region probably does not function as an 'interface recognition site' in the lipases, although it might well bind fatty acid esters of ecdysteroids or single lipid molecules in the vitellogenins. C-terminally of this 106-residue region, a surface loop ('flap') covers the active site. No residue within this loop is conserved through all lipases, but adjacent segments exhibit 60-70% residue identity. Hepatic and lipoprotein lipases probably hydrolyze both soluble and emulsified substrates at the same site. They lack residues corresponding to a second active site postulated in pancreatic lipase to account for hydrolysis of soluble substrates. In addition, due to structural differences the flap could prevent entry of soluble substrate molecules into the active site of pancreatic lipase.

Comparison of the action of lipoprotein lipase on triacylglycerols and phospholipids when presented in mixed liposomes or in emulsion droplets.

We have compared the action of lipoprotein lipase on liposomes of egg yolk phosphatidylcholine containing less than saturating amounts of trioleoylglycerol (less than 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 Pseudomonas fluorescence. 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.