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Lipoprotein lipase (LPL) activity and mass in plasma and their increase after heparin administration were measured in 61 men who had suffered myocardial infarction before the age of 45 years and in 69 population-based age- and sex-matched control subjects without coronary heart disease to study the relations between these parameters in plasma and their correlations with plasma lipoproteins in subjects with a wide range of lipoprotein and LPL levels. There was a relatively large amount of LPL protein compared with LPL activity in preheparin plasma, indicating that the majority of circulating LPL is catalytically inactive. LPL mass and activity in postheparin plasma (postheparin minus preheparin values) were highly correlated, and the calculated mean specific activity (0.35 mU/ng) was in the range expected for catalytically active LPL. Hence, heparin releases mainly active LPL. The four LPL parameters (mass and activity in plasma and their increase after heparin administration) were not related to each other, except for postheparin plasma LPL mass and activity, and they showed different correlations with plasma lipoprotein lipid concentrations. There was a strong positive correlation between LPL mass in preheparin plasma and the HDL cholesterol level as well as weak negative relations to VLDL triglyceride and cholesterol concentrations in the patients. In contrast, preheparin LPL activity showed no correlation with the HDL cholesterol level but weak positive relations to VLDL triglyceride and cholesterol concentrations in the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein lipase has been found to efficiently mediate binding of lipoproteins to cell surfaces and to the low density lipoprotein (LDL) receptor-related protein (LRP) under cell culture conditions (Beisiegel et al. 1991. Proc. Natl. Acad. Sci. USA. 88: 8242-8346). This supports the previously proposed idea that the lipase could have a role in receptor-mediated uptake of chylomicron remnants in the liver. We have investigated the effects of lipoprotein lipase on the clearance of chylomicrons during perfusions of rat livers. The chylomicrons were doubly labeled in vivo with [14C]retinol (in retinyl esters) and with [3H]oleic acid (in triacylglycerols) and were collected from lymph. In the absence of any lipase the clearance of chylomicron label from the perfusion medium was slow. Addition of lipoprotein lipase caused lipolysis of chylomicron triacylglycerols as evidenced by increased levels of 14C-labeled fatty acids in the perfusate. Simultaneously, the level of [14C]retinyl esters in the perfusate decreased dramatically, indicating core-particle removal. Similar effects were seen with an unrelated lipase from Pseudomonas fluorescens. To discriminate between the effects of lipolysis and a true liganding effect of the lipoprotein lipase protein, the active site inhibitors tetrahydrolipstatinR and hexadecylsulfonylfluoride were used to reduce or totally inhibit the catalytical activity. With lipase covalently inhibited by the latter inhibitor, lipolysis during perfusions was low or absent. Nonetheless, the inhibited enzyme had a clear effect on the removal of chylomicrons by the liver. With 1.2 micrograms of inhibited lipase/ml perfusate, about 70% of the core label had been removed after 15 min as compared to about 20% in perfusions without lipase. With identical amounts of active lipoprotein lipase protein, more than 90% of the label was removed. We conclude that any lipase causing lipolysis of chylomicrons can stimulate their clearance by the liver, but that lipoprotein lipase has an additional effect on the removal, which is not dependent on its catalytic activity.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the reverse cholesterol pathway but its role in lipid metabolism is still unclear. We have generated mice transgenic for a 7-kb genomic DNA fragment comprising the 6 exons and 5 introns of the LCAT gene with 1932 bp of 5' flanking and 908 bp of 3' flanking sequences. One line had integrated about 30 copies and expressed about 40-fold increased LCAT activity in a human test system. The expression showed correct tissue specificity of the human LCAT gene. Increased LCAT activity resulted in a decrease of plasma triacylglycerols below 50% of fasting controls. This reduction was seen in all lipoprotein fractions. Lipoprotein lipase activity did not change significantly, whereas hepatic triacylglycerol lipase increased markedly. Plasma total cholesterol was similar in fasting transgenic and control mice, but low-density lipoprotein and very low-density lipoprotein cholesterol were reduced to about 50%. High-density lipoprotein cholesterol increased about 20%, accompanied by a correspondingly increased size and a higher cholesterol efflux-stimulating activity of transgenic LCAT high-density lipoprotein. Both apolipoprotein A-I and A-II plasma concentrations increased in transgenic mice. Plasma triacylglycerol and cholesteryl ester fatty acid distribution showed an increased proportion of palmitic acid, whereas oleic, linoleic and arachidonic acid decreased, thus resembling more closely the human situation. Overexpression of the human LCAT gene provokes major changes in plasma lipoprotein and apolipoprotein concentrations, resulting in a less atherogenic plasma lipoprotein profile through a reduction in atherogenic and an increase in anti-atherogenic lipoproteins.

Heparin-derived deca- and octa-saccharides were subjected to affinity chromatography on lipoprotein lipase-Sepharose and the fractions eluted at high salt concentration were analysed by strong-anion-exchange chromatography. Two high-affinity decasaccharides were isolated and the structure determined by one- and two-dimensional 1H-n.m.r. spectroscopy. The affinities of 3H-labelled low-molecular-mass heparin and size-fractionated deca-, octa-, and hexa-saccharides for lipoprotein lipase immobilized on microtitre plates were determined from saturation curves. From competition experiments the affinities of unlabelled heparins and pure deca- and hexa-saccharide fragments were determined. The binding was size- and charge-dependent, but structural dependency was also indicated. Thus substitution of a 2-O-sulphated L-iduronic acid with D-glucuronic acid was less important than the sulphation pattern of the D-glucosamine residue for affinity for lipoprotein lipase. Heparin inhibits binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein. The effects of size, charge and structure for this inhibition were studied. The ability of the heparin fragments to inhibit binding correlated with their affinity for lipoprotein lipase. This indicates that the inhibition of the binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein by heparin is exclusively mediated by binding of heparin to lipoprotein lipase.

Lipoprotein lipase and hepatic lipase are members of a gene family which also contains pancreatic lipase. High activity of lipoprotein lipase is present in extrahepatic tissues in all mammals studied and also in birds. The activity of hepatic lipase varies more. To investigate the evolutionary relationship, lipase activities in tissues of some lower vertebrates were measured. In fish and in frog, low activities with the characteristics of lipoprotein lipase were found. Serum from frog and from fish, and plasma from chicken, stimulated lipoprotein lipase in vitro, indicating that these species contain analogues to human apolipoprotein C-II. Little or no hepatic lipase-like activity was found in post-heparin plasma or in liver homogenates of chickens. In fish liver, lipase activity with an apparent heparin affinity similar to, or even higher than lipoprotein lipase was found. Frog liver contained a small amount of lipase activity with high heparin affinity. This activity was inhibited both by apolipoprotein C-II and by 1 M NaCl. It is not clear whether the low lipase activities in livers from fish and from frog are variants of hepatic lipase. Since lipoprotein lipase and apolipoprotein C-II are already present in fish, this lipase probably evolved before hepatic lipase.

Cell surface proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. To investigate the organization of these molecules at the cell surface, the distribution of two well-known proteoglycan ligands has been studied. These ligands, lipoprotein lipase and basic fibroblast growth factor, showed a characteristic binding pattern consisting of highly organized parallel arrays that crossed the upper surface of human skin fibroblasts. The proteoglycan nature of the binding sites was evident from their susceptibility to heparinases, and from ligand displacement by heparin. Parallel localization of the ligands and actin, and treatment of the cells with cytochalasin, showed that the binding proteoglycans are organized by the actin cytoskeleton. The ligands induced a different behaviour of the binding sites on incubation of the cells at 37 degrees C. Lipoprotein lipase produced a movement of the binding proteoglycans along the actin filaments towards the cell center. In contrast, after binding of basic fibroblast growth factor the binding proteoglycans remained spread over the cell surface and actin depolymerization was induced. Since an increasing number of ligands appear to depend on proteoglycans for their interactions with their high affinity receptors, distribution and movement of proteoglycans at the cell surface that is organized by the actin cytoskeleton could direct and enhance the encounters between the ligands and their specific receptors.

In this chapter we review recent molecular knowledge on two structurally related mammalian triglyceride lipases which have evolved from a common ancestral gene. The common property of the lipase family members is that they interact with non-polar substances. Pancreatic lipase hydrolyzes triglycerides in the small intestine in the presence of many dietary components, other digestive enzymes and high concentrations of detergents (bile salts). Lipoprotein lipase acts at the vascular side of the blood vessels where it hydrolyses triglycerides and some phospholipids of the circulating plasma lipoproteins. A third member of the gene family, hepatic lipase, is found in the liver of mammals. Also, this lipase is involved in lipoprotein metabolism. The three lipases are distantly related to some non-catalytic yolk proteins from Drosophila (Persson et al., 1989; Kirchgessner et al., 1989; Hide et al., 1992) and to a phospholipase A1 from hornet venom (Soldatova et al., 1993).

It has previously been shown that lipoprotein lipase can mediate uptake of remnant lipoprotein particles via binding to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP). Binding of lipoprotein lipase, and of triglyceride-rich lipoproteins associated with the lipase, to LRP depends on an intact carboxyl-terminal folding domain of the lipase (Nykjaer, A., Bengtsson-Olivecrona, G., Lookene, A., Moestrup, S. K., Petersen, C. M., Weber, W., Beisiegel, W., and Gliemann, J. (1993) J. Biol. Chem. 268, 15048-15055). Here we show that the site for binding to the receptor is within residues 380-425 of the bovine and residues 378-423 of the human lipoprotein lipase. We demonstrate that a carboxyl-terminal fragment of human lipoprotein lipase (residues 378-448), expressed as fusion protein in Escherichia coli, binds to purified and cellular LRP but not to lipoproteins. Binding of the fragment to purified LRP was blocked by heparin. In addition, the fragment inhibited the binding of lipase and the lipase-mediated binding of lipoproteins to the purified receptor. The fragment exhibited reduced binding to proteoglycan-deficient cells. Moreover, the fragment inhibited the uptake of lipoproteins in cells mediated by the lipase via binding to heparan sulfate proteoglycans and LRP. We conclude that the fragment contains the site for binding to LRP and a candidate site for interaction with heparan sulfate proteoglycans, whereas binding to lipoproteins is inefficient. The fragment can therefore inhibit the lipase-mediated lipoprotein uptake, a process that may promote the development of atherosclerosis when occurring in cells of the arterial wall.

The polycation protamine impedes the catabolism of triglyceride-rich lipoproteins and this has been suggested to be due to intravascular inactivation of lipoprotein lipase. We have made intravenous injections of protamine to rats and found that both lipoprotein lipase and hepatic lipase activities were released to plasma. The effect of protamine was more short-lived than that obtained by injection of heparin. The release of hepatic lipase by protamine was as effective as the release by heparin, while the amount of lipoprotein lipase released by protamine was only about one-tenth of that released by heparin. This was not due to inactivation of lipoprotein lipase, since injection of an excess of heparin 10 min after injection of protamine released as much lipoprotein lipase activity to plasma as in controls. The results in vivo differed from those obtained in model experiments in vitro. Protamine was able to almost quantitatively release both lipoprotein lipase and hepatic lipase from columns of heparin-agarose. The displacement was dependent on the total amount of protamine that had passed over the column, indicating that it was due to occupation by protamine of all available binding sites. Our results in vivo showed that the binding sites for lipoprotein lipase were not blocked as efficiently as those for hepatic lipase, indicating that the binding structures were not identical. It was concluded that the impaired turnover of lipoproteins by protamine probably was due to prevention of binding of the lipoproteins to endothelial cell surfaces rather than to impaired lipase function.

This study compares the clearing and metabolism of three different lipid emulsions. They had the same phospholipid emulsifier and similar particle sizes. In one (LLL) the core component was long-chain triglycerides (TG), the second (MMM/LLL) contained equal molar amounts of medium- and long-chain TG, the third (MLM) contained synthetic TG with medium-chain (M) fatty acids in the 1,3-positions and a long-chain (L) fatty acid in the 2-position. In model experiments with bovine lipoprotein lipase, the MMM component was hydrolyzed preferentially in the MMM/LLL emulsion so that the initial products were M fatty acids and M monoglycerides. The MLM emulsion, in contrast, gave M fatty acids and formation of L-MG (monoglyceride) throughout hydrolysis. For in vivo studies [3H]oleic acid was incorporated into the emulsion TG as marker for the long-chain component. After bolus injection to rats, the MMM/LLL and MLM emulsions were cleared more rapidly than the LLL emulsion. This was true at all TG loads studied (4-64 mg for a 200 g rat). The labeled oleic acid was oxidized somewhat more rapidly when administered in the MLM emulsion compared to the MMM/LLL emulsion. There were only slight differences in tissue distribution of label. Hence, differences in in vivo metabolism of the long-chain fatty acids were small compared to the marked differences in TG structure and in patterns of product release during in vitro lipolysis.

A strong candidate for the long-searched CR receptor might be the alpha 2MR/LRP. Presently, we are overseeing a whole series of in vitro experiments from different laboratories that show that LRP expresses all the features for being such a receptor protein. LRP is localized on the liver cell surface, as well as on most other animal cells. It recognizes apo E-enriched lipoproteins as beta-VLDL and CR. There is evidence that CR contain LPL and it has been demonstrated that LPL binds with high affinity to LRP. This has been shown in cell binding experiments with subsequent cross-linking and in direct assays on purified receptor protein. HL, which is expressed in liver cells and localized at the liver cell surface, is also able to bind to LRP. Moreover, LRP is found in endosomes and can mediate the uptake of beta-VLDL and CR. Further studies are necessary to evaluate its role in vivo as well as its regulation. The interplay between the different ligands of this large multifunctional receptor protein needs to be clarified. It should be emphasized here that, by describing LPL as a new mediator of CR uptake in the liver and by providing evidence for a direct interaction between LPL and LRP, the role of LRP in the remnant catabolism has become even more likely.

The influence of cortisol, in the presence of insulin, on the regulation of lipoprotein lipase (LPL) activity was studied in human adipose tissue, using a tissue incubation technique. Tissue pieces were preincubated for 3 days in a control medium containing insulin (7175 pmol/L), then incubated for 2 additional days in the control medium with and without cortisol (1000 nmol/L). After the 5 days of incubation, the levels of LPL messenger ribonucleic acid (mRNA), relative LPL synthesis, and LPL activity (total and heparin releasable) were studied. Cortisol exposure for 2 days increased all of the variables related to LPL. The average increase was 2.5-fold for LPL mRNA, 3.0-fold for relative LPL synthesis, 5.2-fold for total LPL activity, and 9.4-fold for heparin-releasable LPL activity compared to that in controls without cortisol. The results confirm previous findings that cortisol, in the presence of insulin, has a marked stimulatory effect on LPL activity in human adipose tissue in vitro. New data have been presented on the mechanisms of cortisol regulation of LPL activity. They involve both an increased level of LPL mRNA, leading to increased relative LPL synthesis, and additional posttranslational regulation.

Lipoprotein lipase (LPL) was rapidly inactivated by low concentrations of the active-site inhibitor tetrahydrolipstatin (THL). The presence of amphiphils (e.g. long-chain fatty acids) or of lipid/water interfaces (lipid emulsions) was required for inhibition to occur. Apolipoprotein CII increased the maximal inactivation rate constant by 1.8-fold in the presence of an emulsion of long-chain triacylglycerols, but had no effect in the presence of an emulsion of tributyrylglycerol. The fully inhibited enzyme had a ratio of THL/LPL of nearly 2, indicating that both subunits of the LPL homo-dimer bound THL. The THL-LPL complex was stable below pH 7.5. At higher pH reactivation occurred indicating that THL was slowly turned over by the enzyme. The apparent reactivation rate constant was increased about threefold by the presence of lipid/water interfaces. Sucrose density gradient centrifugation revealed that THL induces tetramerisation of LPL. This aggregation was reversible on reactivation of the inhibited enzyme. Binding to heparin was not affected by THL. In contrast, binding to lipid droplets and to lipoproteins was increased, indicating exposure of hydrophobic regions in the inhibited LPL. It is suggested that THL induces local conformational changes in LPL, which may involve opening of the putative surface lid structure which covers the active-site.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In contrast to plasma from other mammals, guinea pig plasma does not stimulate the activity of lipoprotein lipases in vitro. This had led previously to the conclusion that guinea pigs lack an analogue to apolipoprotein CII (apoCII). By adsorption of lipid-binding proteins to lipid droplets, thereby separating them from other plasma components, we could demonstrate apoCII-like activity in guinea pig plasma. On electrophoresis, the CII-like activity co-migrated with one isoform of guinea pig apolipoprotein CIII, identified by amino-terminal amino acid sequence determination (40 residues). By isoelectric focusing in a narrow pH gradient, the activating protein was separated sufficiently from the dominating apoCIII isoform to allow sequence determination of 8 residues from the amino terminus. Six of these were identical to corresponding residues in apoCII from dog and monkey. With the aid of a human apoCII cDNA probe we identified one cross-hybridizing mRNA species (approximately 600 nucleotides) on Northern blots of guinea pig liver. Three positive clones were isolated from a guinea pig liver cDNA library using the same cDNA probe. The nucleotide sequence showed extensive similarities to the previously known human, monkey, and canine sequences, but the signal peptide was 3 amino acid residues longer in the guinea pig protein, and there was a deletion of 4 residues in the putative lipid binding domain. Northern blot analyses indicated that guinea pig apoCII is mainly expressed in the liver with little or no contribution from the intestine.

This study compares a low-Mr heparin preparation with conventional heparin with respect to its interaction with lipoprotein lipase (LPL) in vitro and its effects on the enzyme in vivo. Both heparin preparations were polydisperse in binding to LPL, but on average the low-Mr preparation showed lower affinity. Thus both conventional and low-Mr heparin bound quantitatively to immobilized LPL, and were eluted as broad peaks when a salt gradient was applied, but the peak for low-Mr heparin was shifted towards lower salt concentrations. To displace LPL from immobilized heparin a higher concentration of low-Mr than of conventional heparin was needed. Injection of the low-Mr heparin into intact rats resulted in lower plasma LPL activity than did injection of an equal mass of conventional heparin, but when the liver was excluded from the circulation both heparin preparations resulted in similar plasma LPL activities. In perfused rat hearts, low-Mr heparin had at least the same effect on the release of LPL activity as did conventional heparin. In perfused livers, on the other hand, low-Mr heparin was less effective than conventional heparin in preventing the rapid uptake of exogenous labelled LPL. Hence the apparently lower average affinity of low-Mr heparin for LPL does not result in a demonstrably lower potency to release the enzyme from endothelial binding sites in peripheral tissues, but does result in a substantially decreased effect on the hepatic clearance of the enzyme.

Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase.

Two major isoforms of the bovine analogue to human apolipoprotein (apo) CII were purified from plasma. They were both as effective as human apo CII in activating lipoprotein lipase. Amino acid sequencing revealed that one form contained 79 amino acid residues, and corresponded to human pro apo CII. The other form lacked the first six residues at its N-terminus. This was apparently due to cleavage of the -Gln-Asp- linkage in the sequence H2N-Ala-His-Val-Pro-Gln-Gln-Asp-Glu-, analogous to cleavages described for human apo AI and apo CII. Previous studies with human apo CII have shown that the ability to activate lipoprotein lipase resides in the C-terminal third of the molecule. This was highly conserved in the bovine analogue: of the 30 last residues, 21 are identical. Five residues in this part of human apo CII have been reported to be essential for activation of lipoprotein lipase. Only one of these, Tyr63, is present in the bovine sequence. The bovine structure contains a threonine at position 61, instead of serine in the human, and the four last residues are -Ser-Gly-Lys-Asp instead of the allegedly necessary -Lys-Gly-Glu-Glu. Three differently sialylated isoforms of the bovine analogue to human apolipoprotein CIII were also isolated and partially sequenced. All three lacked the first three N-terminal residues as compared to sequences from other species (man, dog and rat). Sequence differences were more pronounced at the ends than in the central parts of the apo CIII molecules.

This paper presents a case of typical hyperlipoproteinemia type I in a young woman. Her serum triglycerides varied between 2 and 90 mmol/l and she had substantial amounts of apolipoprotein B-48 in fasting plasma. She had no detectable lipoprotein lipase (LPL) activity in post-heparin plasma (less than 0.2 percent of normal). Southern blot analysis suggested no major defect in her LPL gene and Northern blot analysis of adipose tissue RNA showed normal-sized LPL-mRNA. A 2-h [35S]methionine incorporation experiment with adipose tissue pieces in vitro showed that she produced normal-sized LPL and had LPL catalytic activity in the tissue. The amounts were, however, only 5-10% of control. No detectable LPL radioactivity or catalytic activity was released from patient tissue even in the presence of heparin in the incubations. Immunofluorescent staining of adipose tissue biopsies from the patient showed LPL immunoreactivity only in adipocytes and little or none within the capillaries. Treatment of immunoprecipitated labeled LPL with endoglycosidase H showed that the oligosaccharide chains on her enzyme were of the high-mannose type and not processed as in controls. Taken together the data suggest that the patient synthesizes a relatively normal LPL protein which is core-glycosylated and folded into active enzyme as in normal subjects, but is not effectively transported via the Golgi to the cell surface.

To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, we compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of [13C]carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Similar methods with [13C]triolein showed a maximum solubility in phospholipid bilayers of 3 mol % (Hamilton & Small, 1981). Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T1, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, we found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. With increasing concentrations of MCT, saturation was not reached, indicating low affinities of lipase for MCT emulsions, but with LCT emulsion incubated with lipoprotein lipase, saturation was reached at relatively low concentration, demonstrating higher affinity of lipase for LCT emulsions. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. Increasing MCT emulsion amounts had little or no effect on LCT emulsion hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

We have analyzed the binding of lipoprotein lipase (LPL) to the subendothelial extracellular matrix produced by cultured endothelial cells. Binding was linear up to a concentration of 0.5 microgram/ml (10 nM) enzyme used in this study, and equilibrium was achieved after 2 h of incubation with bovine 125I-LPL at 4 degrees C. Heparin and heparan sulfate effectively inhibited the binding of LPL to extracellular-matrix-coated plates; chondroitin sulfate had no effect, while high concentrations of dermatan sulfate or keratan sulfate inhibited binding of LPL to extracellular matrix by only 40%. Basic fibroblast growth factor (bFGF) did not affect LPL binding, while antithrombin-III (AT-III) caused up to a 50% inhibition of enzyme binding to extracellular matrix. alpha-Thrombin. 5.10(-6) M, and its esterolytically inactive derivative, DIP-alpha-thrombin, effectively inhibited binding of LPL to extracellular-matrix-coated plates. alpha-Thrombin was also able to release the extracellular-matrix-bound LPL in an active form. Extracellular-matrix-bound LPL detached into medium containing triolein emulsion and/or serum, and was catalytically active after being released. Extracellular-matrix-bound LPL lost 30% of its activity following incubation at 37 degrees C for 4 h. in contrast to soluble LPL which lost 75% of its activity. It is plausible to conclude from these data that in vivo the subendothelial basement membrane, similarly to extracellular matrix, sequesters and stabilizers LPL secreted into the subendothelial space by non-endothelial cells, and thus may play an important role in determining the route of LPL from its site of synthesis to its site of action.

Lipoprotein lipase (LPL) catalyzes the flux-generating step in transport of fatty acids from lipoprotein triacylglycerols into tissues for use in metabolic reactions. In vitro studies have shown that fatty acids can bind to the enzyme and impede its other interactions. In this study we have searched for evidence of fatty acid control of LPL in vivo by rapid infusion of a triacylglycerol emulsion to healthy volunteers. During infusion the activity of LPL but not of hepatic lipase increased in plasma, but to different degrees in different individuals. The time course for the increase in LPL activity differed from that for triacylglycerols but followed the plasma levels of free fatty acids. This was true during infusions and when the emulsion was given as a bolus injection. In particular there were several instances when plasma triacylglycerol levels were very high but free fatty acids and LPL activity remained low. Model studies with bovine LPL showed that fatty acids displace the enzyme from heparin-agarose. We suggest that in situations when fatty acids are generated more rapidly by LPL than they are used by the local tissue, they cause dissociation of the enzyme from its binding to endothelial heparin sulfate and are themselves released into circulation.

We have previously demonstrated that infusion of Intralipid to rats causes a pronounced increase of the lipoprotein lipase activity in the liver. In this paper we study where in the liver this lipoprotein lipase is located. When isolated livers from Intralipid-treated rats were perfused with heparin, substantial amounts of lipoprotein lipase were released into the perfusate. The identity of the lipase activity was demonstrated by specific inhibition with antisera to lipoprotein lipase, and to hepatic lipase, respectively, and by separation of the two lipase activities by chromatography on heparin-Sepharose. We have also studied the localization of both enzymes by an immunostaining procedure based on post-embedding incubation of ultrathin tissue sections with specific antibodies which were then visualized using protein A-colloidal gold complexes. There was no marked difference in localization for the two enzymes which were both seen at the luminal side of endothelial cells, at the interdigitations of the space of Disse and inside both hepatocytes and endothelial cells. Thus, lipoprotein lipase is present in the liver in positions similar to where the functional pool of hepatic lipase is located and analogous to where lipoprotein lipase is found in extrahepatic tissues. These results raise the possibility that the enzyme has a functional role in the liver.

Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.

We have isolated and sequenced cDNA clones covering the entire coding sequence and short flanking regions of guinea pig lipoprotein lipase. The expression cDNA library used was constructed in lambda gt11 with mRNA derived from adipocytes. The deduced amino acid (aa) sequence starts with a stretch of 17 aa interpreted as a leader peptide. The open reading frame continues with 448 aa residues and ends with a TGA stop codon. Combined with previous data this information allows the assignment of domains in the lipase molecule. A likely candidate for the heparin-binding site is a 9-aa stretch containing five positive charges, analogous to the consensus sequence for receptor-binding sites on apolipoproteins E and B. A previously noted homology to pancreatic lipase is extended. Analysis of polyadenylated RNA from several tissues indicated a high level of expression in adipocytes, heart muscle and mammary gland. No lipoprotein lipase mRNA could be detected in liver. Northern blots revealed three major mRNAs with sizes corresponding to 3.8 kb, 3.3 kb and 2.1 kb, respectively. In adipocytes and heart muscle a fourth mRNA, with an estimated size of 4.5 kb, was also detected. Analysis of genomic DNA by Southern blotting indicated a single gene locus coding for lipoprotein lipase. Hence, modification of the primary transcript seems to be involved in the production of the various mRNAs.

The lipoprotein lipase molecule carries several functional sites. Three of these sites, for interaction with lipid interfaces, with activator protein, and with fatty acids, regulate the action of the enzyme's active site. Another, independent, site on the molecule anchors it to cell surface heparan sulfate and thus holds it in place at the endothelium. Together these properties allow for a fine-tuned regulation of the lipoprotein lipase reaction so that lipids are transported from the right particles, into the right tissues at the right rate.

Combined lipase deficiency (cld/cld) is a recessive mutation in mice which results in massive hyperlipemia and death within 3 days after birth. We studied the effect of this deficiency on lipolytic activities in liver and in pre- and postheparin plasma of mice less than 2 days old. Anti-hepatic lipase serum inhibited more than 85% of the lipolytic activity in liver and plasma of normal newborn mice when assayed in high-salt medium, validating the use of this medium for measuring hepatic lipase activity in mice. Anti-lipoprotein lipase serum, in contrast, inhibited only two-thirds of the lipolytic activity in liver and plasma when assayed in serum low-salt medium, and anti-hepatic lipase serum inhibited the rest. This indicates that assay with serum low-salt medium alone is not specific for lipoprotein lipase activity in mice. Therefore, immunoinhibition was used, as needed, for measuring lipoprotein lipase activity. The livers of unaffected newborn mice contained high levels of both hepatic and lipoprotein lipase activities, 228 and 187 mU/g, respectively. The plasma of unaffected mice contained a high level of hepatic lipase activity, 244 mU/ml, but practically no lipoprotein lipase activity. Heparin injected intraperitoneally increased plasma lipoprotein lipase activity to 152 mU/ml, but had no effect on plasma hepatic lipase activity, in unaffected mice. Hepatic lipase activity was virtually absent from both liver and plasma of cld/cld mice. Lipoprotein lipase activity was present in the liver at a surprisingly high level, 40% of that in normals, but was barely detectable in plasma. Heparin injection increased plasma lipoprotein lipase activity in cld/cld mice, but the increment was less than 10% of that in unaffected mice. Heparin had no significant effect on plasma hepatic lipase activity in defective mice. These findings confirm preliminary observations that hepatic lipase activity in liver and plasma and lipoprotein lipase activity in plasma are markedly reduced in combined lipase deficiency. The unexpected high level of lipoprotein lipase activity in liver of cld/cld mice suggests that regulation of lipoprotein lipase activity in liver of neonatal mice is different from that in other tissues.

Lipoprotein lipase and hepatic lipase were measured in rat plasma using specific antisera. Mean values for lipoprotein lipase in adult rats were 1.8-3.6 mU/ml, depending on sex and nutritional state. Values for hepatic lipase were about three times higher. Lipoprotein lipase activity in plasma of newborn rats was 2-4-times higher than in adults. In contrast, hepatic lipase activity was lower in newborn than in adult rats. Following functional hepatectomy there was a progressive increase in lipoprotein lipase activity in plasma, indicating that transport of the enzyme from peripheral tissues to the liver normally takes place. Lipoprotein lipase, but not hepatic lipase, increased in plasma after a fat meal. An even more marked increase, up to 30 mU/ml, was seen after intravenous injection of Intralipid. Plasma lipase activity decreased in parallel with clearing of the injected triacylglycerol. 125I-labeled lipoprotein lipase injected intravenously during the hyperlipemia disappeared somewhat slower from the circulation than in fasted rats, but the uptake was still primarily in the liver. Hyperlipemia, or injection of heparin, led to increased lipoprotein lipase activity in the liver. This was seen even when the animals had been pretreated with cycloheximide to inhibit synthesis of new enzyme protein. These results suggest that during hypertriglyceridemia lipoprotein lipase binds to circulating lipoproteins/lipid droplets which results in increased plasma levels of the enzyme and increased transport to the liver.

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.