Gunilla Bengtsson

Immunochemical properties of lipoprotein lipase: Development of an immunoassay applicable to several mammalian species

The reaction of bovine lipoprotein lipase with its antibodies was found to be conformation-dependent. One aspect of this was that most antisera were more reactive with denatured than with native 125I-labeled lipoprotein lipase. Another aspect was that denatured lipase did not compete effectively with native lipase for those antibodies which caused inhibition of the enzymes activity. This latter observation leads to the conclusion that the inhibiting antibodies recognize conformation-dependent determinants on the native enzyme. Fab fragments prepared from an inhibiting antiserum blocked the binding of the lipase to triacylglycerol/phospholipid droplets. This suggests that the inhibition results from reaction of the antibodies with the enzyme as it exists in solution, either covering the lipid-binding site on the enzyme or making it impossible for the enzyme to go through the conformational transitions necessary for binding to lipid. Most rabbit antisera did not react with rat or mouse lipoprotein lipase, but some sera showed a weak cross-reaction. Antisera raised in hens showed a much stronger cross-reaction, enough to be useful for heterologous immunoassays. An immunoassay for the bovine lipase was developed. For reproducible results it was necessary to have tracer, standard and samples in denatured form. This was accomplished by heating them in SDS, and running the immunoreaction in a Triton X-100-containing medium.

The hepatic heparin releasable lipase binds to high density lipoproteins

Received 22 August 1980 1. Introduction Injection of heparin releases two lipases into the circulating blood. One, lipoprotein lipase, originates from extrahepatic tissues [ I]. The physiological role of this lipase is to hydrolyze triacylglycerols and phos- pholipids in chylomicrons and very low density lipo- proteins [ 11. The other lipase comes from the liver [2]. It is generally assumed that also this lipase has an important role in lipoprotein metabolism, but it is now known what its physiological substrate is. We demonstrate here binding of thislipase to high density lipoproteins. 2. Materials and methods Human postheparin plasma was obtained from male donors 10 min after intravenous injection of 100 I.U. heparin/kg body weight. The lipase of hepatic origin was partially purified by adsorption of the postheparin plasma with heparin-Sepharose [3]. It was further purified by adsorption to heparin which had been modified by partialN-desulfation followed by acetyla- tion of exposed amino groups [4]. This step removed antithrombin from the lipase preparation. The specific activity of a typical preparation was 50 pm01 fatty acids released/min X mg (pH 8.5,25’C) against a gum arabic stabilized emulsion of [3H] trioleoylglycerol. Human high density lipoproteins were prepared by ultracentrifugation of normal plasma. Very low density lipoproteins and low density lipoproteins were first flotated by centrifugation at d 1.063 g/cm3 for 24 h at 36 000 rev./min in a Beckman Ti 50 rotor, 15°C. High density lipoproteins were then prepared by cen- trifugation of the infranatant at d 1.21 g/cm3 for 48 h at 36 000 rev./min. In one experiment a subfraction of high density lipoproteins isolated in the density 290 range 1.125-l .21 g/cm3 was used (high density lipo- proteins3). The lipoproteins were dialyzed against 0.1 M NaCl 10 mM Tris/Cl pH 8.5. The concentration of high density lipoproteins used in the experiments is expressed as protein determined by the Lowry method. High density lipoprotein-Sepharose was prepared by mixing CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden) with high density lipoproteins (10 mg protein/ml gel) in 0.1 M NaHC03, 0.5 NaCl at 4°C over night. Remaining activated sites on the Sepharose were blocked by 0.1 M ethanolamine (pH 8). The gel was washed with the carbonate buffer, then with 0.1 M acetate pH 4 and stored in 10 mM phosphate, 0.1 M NaCl, pH 7.4 with 0.2% sodium azide. All experiments were carried out within two weeks after the preparation of the gel. Unsubstituted Sepharose 4B was used as a control. Human apolipo- protein CIIIz was prepared from human very low density lipoproteins as described [5]. Intralipid 10% containing [ 14C] oleic acid-labeled trioleoylglycerol was donated by AB Vitrum, Stockholm, Sweden. The triacylglycerol-rich particles were separated from the excess phospholipids by flo- tation through 20 mM Tris/Cl, 0.1 M NaCl, pH 8.5 by centrifugation for 20 min in a Beckman SW-50: 1 rotor at 25 000 rev./min [5]. The top layer was recovered and dispersed in buffer. Albumin was a fraction V preparation from Sigma, St. Louis, MO., USA. The release of labeled fatty acids was determined as described [5]. Deoxycholate was from Merck, Darmstadt, F. R. G. 3. Results The activity of the hepatic heparin-releasable lipase against a triacylglycerol emulsion was strongly inhib-

Rapid removal to the liver of intravenously injected lipoprotein lipase

Lipoprotein lipase was purified from bovine milk and labeled with 125I. After intravenous injection to rats the labeled lipase rapidly disappeared from the blood. The initial half-life was about 1 min and more than 70% of the radioactivity was found in the liver at 10 min. 30 min after the injection about 10% of the injected radioactivity was present in acid-soluble form in blood, indicating that the enzyme had been rapidly degraded. Injection of asialofetuin, ribonuclease B or mannan in amounts known to block the hepatic receptors for glycoproteins with exposed galactose, N-acetylglucosamine or mannose residues did not retard the removal of the lipoprotein lipase. Thus, some other, as yet undefined, receptor is implicated. Lipoprotein lipase is known to bind to heparin and some related polysacchrides. Heparin injected before the enzyme delayed its removal and heparin injected after the enzyme caused an immediate increase in blood radioactivity, signifying return from tissues to blood of labeled enzyme. Lipoprotein lipase is present at the endothelium in several extrahepatic tissues and is rapidly turned over. Its presence in blood in appreciable amounts would cause a derangement of lipid transport. The efficient hepatic removal of the enzyme may thus serve an important physiological purpose in keeping the blood levels of this enzyme low.

Apolipoprotein CII enhances hydrolysis of monoglycerides by lipoprotein lipase, but the effect is abolished by fatty acids.

Lipoprotein lipase is an enzyme that hydrolyzes triglycerides and phospholipids in lipoproteins. Several aspects of its mode of action are not well understood. One is why fatty acids strongly inhibit the activity [ 11. Another is why monoglycerides do not accumulate during hydrolysis of triglycerides, although the rate of monoglyceride hydrolysis has been reported to be substantially lower than the rate of triglyceride hydrolysis [2-41. We report here that under appropriate experimental conditions monoglyceride hydrolysis is enhanced several-fold by apolipoprotein CII and reaches rates exceeding those for triglyceride hydrolysis. We also demonstrate that fatty acids interfere with and at higher concentrations completely abolish the lipolysis-promoting effects of the cofactor protein. This previously unrecognized effect of fatty acids must be a major factor in their inhibition of acylglycerol hydrolysis.

Purification and properties of lipoprotein lipase in guinea pig milk

Lipoprotein lipase was purified from guinea pig milk by chromatography on heparin-Sepharose followed by chromatography on an immobilized preparation of heparin that had been N-desulphated and then acetylated. This second step was necessary to separate a plasma protein, presumably antithrombin, from the lipase. The guinea pig enzyme turned out to be quite similar to lipoprotein lipase from bovine milk with respect to composition and molecular size. Furthermore, the specific activities and the dose-response relations for activation by apolipoprotein C-II were quite similar for the two enzymes. Antibodies raised against the guinea pig milk enzyme inhibited not only this enzyme but also the lipoprotein lipase activity in post-heparin plasma and in homogenates from adipose tissue and heart.

Activation of lipoprotein lipase by apolipoprotein CII. Demonstration of an effect of the activator on the binding of the enzyme to milk-fat globules.

Lipoprotein lipase catalyses the hydrolysis of triacylglycerols and phospholipids in chylomicrons and very low density lipoproteins. This enables transport of fatty acids and of monoacylglycerols from the lipoproteins into the tissue cells [ 11. From in vitro experiments it is known that apolipoprotein CII, which is a constituent of these lipoproteins, is an activator for lipoprc;t.ein lipase [2,3]. That this activator has a function also in vivo is demonstrated by the fact that patients deficient in apolipoprotein CII accumulate large amounts of chylomicron-like lipoproteins in their blood [4]. The development of methods to purify both the lipase and the activator protein has made it possible to study the mechanism of activation in delined model systems. In these systems lipoprotein lipase usually has significant activity even in the absence of its activator, and the activator causes a < IO-fold stimulation of the reaction rate [3,5-121. Studies using Intralipid [6], monolayers of diacylglycerols [ 111, triacylglycerol-coated gjass beads [ 131, apolipoprotein CII-deficient very-lowdensity lipoproteins [14] and liposomes of dipalmitoyl phosphatidyl choline [ 151 have demonstrated that lipoprotein lipase binds to lipid-water interfaces. In these systems, the activator protein is not needed for the binding, and it has therefore been concluded that the activator acts after the enzyme has bound to the interface [6,11,14]. This view is supported by experiments with non-lipid binding fragments of apolipoprotein CII which under certain circumstances activate the enzyme [9,10,14,16]. There are, however, some studies which indicate that the activator can also affect the binding of the lipase. Fielding [ 171, as well as Posner and Morrison [ 181 have reported that a mixture of apolipoproteins, including the activator, lowers the apparent Km of lipoprotein lipase for the lipid substrate. Schrecker and Greten found a lower apparent Km for triacylglycerol droplets stabilized by apolipoprotein CII than for droplets covered by CI or CIII, and concluded that this was because CII enhanced the binding of the lipase to the lipid droplets [19]. Matsuoka et al. [20], Fitzharris et al. [21] and Catapano et al. [ 141 found in studies with CII-deficient very-low-density lipoproteins, that the main effect of CII is to lower the apparent K,,, of lipoprotein lipase for its lipoprotein substrate. Since both lipoprotein lipase and apolipoprotein CII are lipid binding proteins and since a direct protein-protein interaction between lipoprotein lipase and its activator has been implicated by several studies [22-241, it would be expected that one protein could affect the binding of the other under certain circumstances. These considerations suggest that the full effect of CII is not seen in those model systems where the enzyme itself binds well to the lipid droplets. It was known that milk-fat globules are almost completely resistent to lipolysis by lipoprotein lipase as long as their structure is intact; large amounts of lipoprotein lipase are present in bovine milk but little or no lipolysis takes place [25]. Only small amounts of this lipase

Interaction of heparin with proteins Demonstration of different binding sites for antithrombin and lipoprotein lipase

Heparin is a polysaccharide with unique biological effects. It impedes the coagulation of blood by accele- rating the reaction between antithrombin (a protease inhibitor in plasma) and a number of the proteolytic enzymes operating in hemostasis [ 1,2] . Furthermore, heparin has the ability to release lipases from tissue sites into the circulating blood (for review see ref. [3]). The antithrombin-stimulating effect of heparin pre- sumably depends on specific binding between the polysaccharide and the protein, resulting in a confor- mational change in the inhibitor molecule [4,5 ] . The mechanism of lipase release is unclear but probably involves the formation of heparin-lipase complexes

Immunohistochemical localization of lipoprotein lipase in human adipose tissue

The distribution of lipoprotein lipase (LPL) was studied in needle biopsies of human adipose tissue. Antibodies against bovine milk LPL react with and inhibit the activity of the human enzyme. These antibodies were used for immunohistochemical studies of the distribution of LPL in human adipose tissue. Immunoreactive enzyme was observed in adipocytes and connective tissue cells resembling preadipocytes. It was also seen in perivascular cells, in capillaries and in larger vessels. Intravenous administration of heparin led to a substantial decrease of immunodetectable LPL in vessels, whereas the enzyme in adipocytes and connective tissue cells was unaffected.

Binding of deoxycholate to lipoprotein lipase

Abstract Binding of deoxycholate to lipoprotein lipase from bovine milk was demonstrated by equilibrium dialysis and by charge shift electrophoresis. The detergent increased the solubility and stability of the enzyme in aqueous buffers, but did not change its elution position on gel filtration. It is concluded that each molecule of enzyme binds several molecules of deoxycholate without denaturation. This signifies the presence of a lipid-binding region in the native structure of the enzyme.

The effects of pH and salt on the lipid binding and enzyme activity of lipoprotein lipase

This paper demonstrates a striking difference between the effects of salt and pH on the activity of lipoprotein lipase against two different substrates: Intralipid and bovine milk fat droplets. With the former substrate 1 M NaCl caused only a slight reduction in enzyme activity and the stimulation by apolipoprotein C-II was the same from 0.1 to 1.1 M NaCl. In contrast, 0.5 M or more NaCl virtually abolished the enzyme activity in the milk system. In this system the salt also abolished binding of the enzyme to the lipid droplets, whereas in the Intralipid system most of the enzyme remained bound even at 1 M NaCl. A similar picture was obtained with respect to effects of pH. In the milk system the activity decreased sharply at pH values above 8.5, whereas in the Intralipid system it continues to rise to pH 10, and the stimulation by activator protein is the same at all pH values. Correlating with this, the binding of the enzyme to the lipid droplets was highly dependent on pH values in the milk systems, with optimum binding around pH 8, whereas in the Intralipid system most of the enzyme remained bound to the lipid droplets at all pH values. These studies demonstrate that apolipoprotein C-II can activate lipoprotein lipase at a wide range of salt concentrations and of pH. They suggest that the well-known effects of high salt concentrations and of high pH to decrease lipoprotein lipase activity are exerted primarily on the enzyme itself.