Partial purification of locust flight muscle lipoprotein lipase (LpL): apparent differences from mammalian LpL

Comp. Biochem. Physiol. Vol. 88B, No. 2, pp. 523-527, 1987

0305-0491/87 $3.00+ 0.00 © 1987PergamonJournals Ltd

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PARTIAL PURIFICATION OF LOCUST FLIGHT MUSCLE LIPOPROTEIN LIPASE (LpL): APPARENT DIFFERENCES FROM MAMMALIAN LpL M. C. VAN HEUSDEN, D. J. VAN D ~ HOP.ST, J. M. VAN DOORN and A. M. TH. BEENAKKERS Department of Experimental Zoology, University of Utrecht, 8 Padualaan, 3508 TB Utrecht, The Netherlands (Tel.: 030-533084) (Received 22 December 1986)

Abstract--1. An attempt was made to purify llpoprotein lipase (LpL) from the flight muscle of the migratory locust based on affinity for heparin, which is known to avidly bind mammalian LpL. 2. However, locust LpL appeared to completely lack this property, which indicates that the suggested membrane-binding of locust LpL is very different from that of mammalian LpL: a heparin-like glycosaminoglycan is not involved. 3. Since locust LpL lacks heparin affinity, other purification methods were assayed. Solubilization of locust LpL was obtained by the detergent Tween 20. 4. Though both anion and cation exchange chromatography resulted in the complete loss of enzyme activity, partial purification of locust LpL was achieved by gel filtration chromatography.

INTRODUCTION The migratory locust is known to rely mainly on diacylglycerol (DG) as a fuel for prolonged flight (for a review see Beenakkers et al., 1985). This D G is transported in the haemolymph by lipophorins and is hydrolyzed by the flight muscles, yielding fatty acids and glycerol (Van tier Horstet al., 1983). Whereas the fatty acids are further oxidized within the flight muscle cells to generate the energy required for wing movement, most of the glycerol diffuses to the fat body where it is mainly reincorporated in DG. Wheeler et al. (1984) showed the presence of lipoprotein-specific lipase activity in locust flight muscle homogenates, using 14C-glycerol-labelled natural lipoproteins as a substrate. Compared to the high density lipophorin (HDLp) Ay~ow (Ay) (which transports D G in the resting insect), the low density lipophorin (LDLp) A + (which is formed during flight by association of lipophorin Ay with the small glycoprotein C2 and D G mobilized from fat body; see review Beenakkers et al., 1985) appeared to be by far the better substrate for the flight muscle lipoprotein lipase (LpL) (Wheeler and Goldsworthy, 1985; Van Heusden et al., 1986). In addition to these data on substrate specificity, a limited characterization of the enzyme in a crude flight muscle preparation has been reported (Wheeler et al., 1984) and indications for the location of the LpL in the plasma membrane have been obtained (Wheeler and Goldsworthy, 1985). However both the mechanism of D G hydrolysis at the flight muscles and the molecular and kinetic properties of the LpL are still largely unknown. By contrast, in the last decade there has been a marked increase in knowledge on mammalian LpL. Molecular and kinetic properties, including the activation of the enzyme by apoprotein C-II, have been studied extensively (for a review see Quinn et al., 1982). Furthermore, the LpL has been localized on the endothelial membrane, loosely bound to heparan

sulphate, a glycosaminoglycan (Cheng et al., 1981). A distinguishing characteristic of mammalian LpL is its affinity for heparin (Olivecrona et al., 1977). Since affinity for heparin is higher than for heparan sulphate (Bengtsson et al., 1980), injected beparin would compete for the heparan sulphate binding site on the LpL and detach the enzyme from the endothelial surface. A purification procedure, based on this heparin affinity, is routinely used (for a review see Smith and Pownall, 1984). Therefore, in this paper an attempt was made to purify the locust flight muscle LpL by heparin affinity chromatography. Since locust LpL appeared to lack any affinity for heparin, other isolation procedures were performed, resulting in a partial purification of the enzyme. MATERIALSAND METHODS Insects Locusta migratoria were reared under crowded conditions as described previously (Van Heusden et al., 1984). Adult

male insects (12-14 days after imaglnal ecdysis) were used in all experiments. Lipoprotein lipase assay

Locust LpL activity was measured with the total protein fraction of haemolymph, containing the flight-specific lipophorin A +. In vivo labelling of the iipophorin in the DG-moiety with 14C-glycerolhas been described previously (Van Heusden et al., 1986). Aliquots of 30/~1 of enzyme preparation were incubated with 70/zl of substrate preparation, for 30 rain at 35°C. The reaction was stopped by the addition of 0.5 % trichloracetic acid (TCA). Radioactivity in the TCA-supernatant, containing the released glycerol, was measured in Emulsifier Scintillator 299 TM (Packard) using a fiquid scintillator spectrometer model 4450 (Packard). For comparison, the activity of purified rat LpL was assayed, according to the method of Nilsson-Ehle and Schotz (1976) using as a substrate 3H-olcic acid labelled trioleoylglycerol, emulsified with phosphatidylcholine, to which serum (containing apoprotein C-II, the activator of LpL) was added. Incubations of 0.1 ml of enzyme prepara523

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M. C. VAN HEUSDENet al.

tion with 0.1 ml of substrate emulsion, for 30 rain at 30°C, were stopped by the addition of 3.25 ml of methanolchioroform-heptane 1.41:1.25:1 (v/v/v), followed by 1.05ml of 0.1M potassium carbonate-borate buffer (pH 10.5). After vigorous mixing and centrifuging (15 rain at 3000g), radioactivity was measured in the methanol-water upper phase, as described above.

Enzyme preparation Dorso-longitudinal locust flight muscles were dissected, rinsed on ice in incubation buffer (28 mM Na2I-IPO4, 5 mM NaH2PO 4, 81raM NaC1, 14raM KCI, 4.3ram MgCIv 4.1 mM NaHCO3, pH 7.65 to which 0.02% NaN3 was added, according to Wheeler et al., 1984), blotted on tissue paper and sonicated (on ice, three times 5 see) at a concentration of 1-3 pairs of muscles per ml of incubation buffer. To investigate the effect on the solubility of the LpL, heparin (5000 U/ml, Organon, Oss, The Netherlands) was added to a sonicated homogenate at a concentration of 100 or 200 U/ml homogenate, which was subsequently stirred for 30 rain on ice or at room temperature and centrifuged at 30,000 g (60 rain, 4°C). The effect of the non-ionic detergent Tween 20 (Sigma) on solubility of the LpL was tested by adding the detergent to the incubation buffer prior to sonication of the muscle tissue, at concentrations ranging from 0.1 to 1% (v/v) and LpL activity was measured after stirring for 60 rain at room temperature and centrifuging at 30,000 g (60 rain, 4°C). Isolation of LpL by column chromatography was assayed with enzyme preparations delipidated by an acetone-ether extraction. Dissected and rinsed muscles were sonicated in acetone (on ice), washed with cold acetone and with cold diethylether and dried. The acetone-ether powder was dissolved by sonication in incubation buffer containing 0.5% Tween 20. The 30,000 g supernatant was used for column chromatography. Rat LpL was purified from postheparin plasma, collected 5-10 rain after injection of heparin (100 U/kg rat).

Purification procedures Heparin affinity chromatography was carried out on heparin-Ultrogel A4 (LKB). The 30,000g supernatant (approx. 20rag protein) of a delipidated and Tween 20-treated flight muscle homogenate was applied to a heparin-Ultrogel A4 column (10 x 1 era), which was subsequently eluted with incubation buffer until A ~ had decreased to zero. Bound proteins were eluted with a gradient from 81 mM to 3 M NaC1 (60 ml) and collected fractions were assayed for LpL activity. For the purification of rat LpL, 5 ml of rat post-heparin plasma was applied to the same column, eluted with incubation buffer and subsequently with a NaCI gradient as described above. Gel filtration chromatography was performed on Ultrogel AcA22 (LKB), Biogel P200 (Bio-Rad) or Sephaeryl $200 (Pharmacia). All columns (100 × 1.6 era) were equilibrated and eluted with incubation buffer containing 0.5% Tween 20, at a flow rate of 6 ml/hr. Ion-exchange chromatography was performed on a DEAE-Sephacel column (Pharmacia) (15 × 1 era) equilibrated in 0.01 M phosphate buffer pH 7.65 and eluted with a gradient from 0.01 to 0.5M NaC1 (60m l), or on a CM~epharose CL-6B column (Pharmacia) (15 × 1 era) equilibrated in 0.005 M phosphate buffer pH 6.0 and eluted with a gradient from 0.005 to 0.4 M NaCI (60 ml).

RESULTS

Solubilization of the LpL When heparin was added to a crude flight muscle homogenate at concentrations of 100 and 200 U/ml, heparin had no influence on either total LpL activity

or the partition of the LpL activity between supernatant and sediment. The non-ionic detergent Tween 20, however, when added to the incubation buffer prior to homogenization of the muscle tissue, did solubilize the LpL. In the absence of detergent, LpL was shown to be pelleted (for 80%) by low speed centrifugation (Van Heusden et al., 1986). Upon the addition of Tween 20 and stirring for 60rain at room temperature, the enzyme activity shifted to the supernatant, the effect being maximal (80% of the LpL activity in the supernatant) at a concentration of 0.5% Tween 20.

Affinity chromatography The supernatant of a delipidated and Tween 20-treated flight muscle homogenate was applied to a heparin Ultrogel A4 column. LpL activity quantitatively eluted from the column without binding to heparin (Fig. IB). As a control, rat post-heparin plasma was applied to the same column. Both hepatic lipase and extrahepatic LpL bound to the Ultrogelcoupled heparin and were eluted with a salt gradient (Fig. 1A).

Other purification procedures Of the several gel filtration media assayed, Ultrogel AcA22 resulted in the best purification yield of the LpL. Whereas loss of enzyme activity occurred on the other gel filtration media, on Ultrogel AcA22 the enzyme was eluted quantitatively (Fig. 2). However, since elution fractions containing the LpL activity largely coincided with a broad protein peak, purification yields of not more than 4 times were reached. At concentrations of Tween 20 higher than 0.5%, the LpL activity peak shifted to the void volume, probably as a result of micelle formation by excess detergent. Both anion and cation exchange chromatography were assayed for purification of the LpL from the 30,000g supernatant of a delipidated and Tween 20-treated flight muscle homogenate, or from the elution fractions of AcA22 gel filtration containing the partially purified LpL activity. However, both procedures resulted in the complete loss of enzyme activity.

Substrate specificity Locust LpL activity in a total muscle homogenate was assayed with the rat serum activated trioleoylglycerol substrate used for the rat LpL. No lipolytic activity could be measured. When rat serum as activating agent (containing apoprotein C-II) for the rat LpL was replaced by centrifuged (12,000 g, 3 rain) haemolymph of resting locusts, still no locust LpL activity could be measured. DISCUSSION Mammalian LpL is localized on the luminal surface of capillary endothelial cells (for a review see Cryer, 1981). Hydrolysis of very low density lipoprotein (VLDL)- and chylomicron-triacylglycerol (TG) takes place in the capillary lumen; the resulting fatty acids are transported through the endothelial layer and taken up into the tissue by a mechanism still to be elucidated (Scow et al., 1980). Since insects lack

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a circulatory system, the organs bath directly in the body fluid: only a permeable connective tissue matrix separates the flight muscle cells from the haemocoel (Ashurst, 1985). Diacylglycerol (DG), the main substrate for the locust flight muscle during sustained flight, is transported in the haemolymph by rest- or flight-specific lipophorins (for a review see Beenakkers et al., 1985) and is hydrolyzed at the flight muscle by a lipoprotein-specific lipase (Wheeler et al., 1984). The flight-specific LDLp A +, carrying large amounts of DG, has been shown to be the preferred substrate for the flight muscle lipoprotein lipase (LpL) (Wheeler and Goldsworthy, 1985; Van Heusden et aL, 1986). Recent data on immunocytochemical localization of lipophorins in the flight muscle show that, both at rest and during flight, the

lipophorins are restricted to the extracellular matrix (Van Antwerpen et ai., 1987). These data suggest the hydrolysis of lipophorin-carried DG to occur extracellularly, a hypothesis which is further supported by the recently described shuttle function of the flight-specific lipophorin: protein components are released upon DG hydrolysis at the flight muscle and are reused in further transport of DG (Van Heusden et ai., 1987). Extracellular hydrolysis of DG would necessitate the LpL to be localized in the plasma membrane or in the extracellular matrix. Sedimentation of the LpL activity by low speed centrifugation indicates the insolubility of the enzyme. Most of this pelleted LpL activity was recovered in the supernatant after treatment of the homogenate with the non-ionic detergent Tween 20. These results agree

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with the hypothesis that the LpL is localized in the plasma membrane or indirectly linked to it. The observations of Wheeler and Goldsworthy (1985), who described high specific activity of the LpL in a plasma membrane-containing fraction, obtained by ultraeentrifugation, concur with this hypothesis as well. For mammalian LpL, heparin affinity chromatography is a widely used method to purify the enzyme (for a review see Smith and Pownall, 1984). In contrast, addition of heparin to a crude flight muscle homogenate had no effect on the solubility of the LpL: the enzyme was still pelleted at low speed eentrifugation. The inability of heparin to displace the locust LpL from its membraneous environment indicates that locust LpL has no affanity for beparin. This was supported by experiments involving heparin affinity chromatography. Although LpL and hepatic lipase from rat postheparin plasma were bound to heparin Ultrogel A4, locust flight muscle LpL eluted from the column without binding. These results demonstrate that the suggested membrane-binding of locust LpL is clearly different from that of mammalian LpL: a heparin-like glycosaminoglycan is not involved. Therefore, purification of the enzyme was further attempted using gel filtration and ion-exchange chromatography. Enzyme activity was lost during ionexchange chromatography. Although the enzyme retained its activity on Ultrogel AcA22 and was eluted with a yield of 100%, only a slight purification was obtained with this method. Measurements of locust LpL activity were performed with its natural substrate: the flight-specific low density lipophorin which was labelled with ~4C-glyeerol in vivo. However, the specific radioactivity of the substrate is limited, while the preparation of large m o u n t s of lipophorin is laborious. Therefore the availability of a glycerol-labelled

artificially prepared lipid substrate, which is hydrolyzed by the locust flight muscle LpL, would be an advantage for studying a further purification procedure for the enzyme. The TG-substrate emulsion used as a substrate for mammalian LpL was, however, not hydrolyzed by the locust LpL, indicating that the enzyme displays substrate specificity. Further experiments are required to define whether the enzyme is able to hydrolyze emulsified D G or whether its lipolytic activity is restricted to lipophorin-bound D G as a substrate. Acknowledgements--The authors wish to thank R. Lam-

mers and Dr H. Jansen (Erasmus University, Rotterdam) for the supply of rat post-heparin plasma, rat serum and the ~H-trioleoylglycerol substrate emulsion, as well as for their assistance in the purification of the rat LpL by heparin affinity chromatography. The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization of Pure Research (ZWO). REFERENCES Ashurst D. E. (1985) Connective tissues. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 249-287. Pergamon Press, Oxford. Beenakkers A. M. Th., Van der Horst D. J. and Van Marrewijk W. J. A. (1985) Insect lipids and lipoproteins, and their role in physiological processes. Prog. Lipid Res.

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Lindahl U. (1980) Interaction of lipoprotein lipase with native and modified heparin-like polysaccharides. Biochem. J. 189, 625-633. Cheng C. F., Oosta G. M., Bensadoun A. and Rosenberg R. D. (1981) Binding of lipoprotein lipasc to endothelial cells in culture. J. biol. Chem. 256, 12893-12898. Cryer A. (1981) Tissue lipoprotein lipase activity and its action in lipoprotein metabolism. Int. J. Biochem. 13, 525-541.

Locust lipoprotein lipase Nilsson-Ehle P. and Schotz M. C. (1976) A stable, radioactive substrate emulsion for assay of lipoprotein lipase. J. Lipid Res. 17, 536-541. Oliveerona T., Bengtsson G., Marklund S. E., Lindahl U. and H66k M. (1977) Heparin-lipoprotein lipase interactions. Fed. Proc. 36, 60--65. Qulnn D., Shirai K. and Jackson R. L. (1982) Lipoprotein lipase: mechanism of action and role in lipoprotein metabolism. Prog. Lipid Res. 22, 35-78. Scow R. O., Blanchette-Mackie E. J. and Smith L. C. (1980) Transport of lipid across capillary endothelium. Fed. Proc. 39, 2610-2617. Smith L. C. and Pownall H. J. (1984) Lipoprotein lipase. In Lipases (Edited by Borgstrom B. and Brockman H. L.), pp. 263--305. Elsevier, Amsterdam. Van Antwerpen R., Linnemans W. A. M., Van der Horst D. J. and Beenakkers A. M. Th. (1987) Cell Tissue Res. submitted. Van der Horst D. J., Abbink J. H. M., Van Doom J. M., Van Marrewijk W. J. A. and Beenakkers A. M. Th. (1983) Glycerol dynamics and metabolism during flight of the locust, Locusta migratoria. Insect Biochem. 13, 45-55.

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Van Heusden M. C., Van der Horst D. J. and lkenakkers A. M. Th. (1984) In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Locusta migratoria. J. Insect Physiol. 30, 684-693. Van Heusden M. C., Van der Horst D. J., Van Doom J. M., Wes J. and Ikenakkers A. M. Th. (1986) Lipoprotein lipase activity in the flight muscle of Locusta migratoria and its specificity for haemolymph lipoproteins. Insect Biochem. 16, 517-523. Van Heusden M. C., Van der Horst D. J., Voshol J. and Bccnakkers A. M. Th. (1987) The recycling of protein components of the flight-specific lipophorin in Locusta migratoria. Insect Biochem. (in press) Wheeler C. H., Van der Horst D. J. and Beenakkers A. M. Th. (1984) Lipolytie activity in the flight muscles of Locusta migratoria measured with haemolymph lipoproteins as substrates. Insect Biochem. 14, 261-266. Wheeler C. H. and Goldsworthy G. J. (1985) Specificity and localization of lipoprotein lipase in the flight muscles of Locusta migratoria. Biol. Chem. Hoppe-Seyler 366, 1071-1077.