Isolation and characterization of lipophorin from Drosophila melanogaster larvae.код для вставкиСкачать
Archives of insect Biochemistry and Physiology 8:243-248 (1988) Isolation and Characterization of Lipophorin From Drosophila mehogaster Larvae Germain J.P. Fernando-Warnakulasuriyaand Michael A. Wells Department of Biochemistry, Biosciences West, University of Arizona, Tucson The hemolymph lipoprotein lipophorin has been isolated from third-instar Drosophila melanogaster larvae by a technique that involves homogenization of whole larvae in a medium containing protease inhibitors and purification of the lipoprotein by density gradient centrifugation. Drosophila lipophorin has a density of 1.16 g/ml and is composed of 62.5% protein, 23.1% phospholipid, 7.4% diacylglycerol, 5.4% triacylglycerol, 0.9% hydrocarbon, and 0.7% sterol. As is the case with other insect lipophorins, Drosophila lipophorin contains two apolipoproteins, apolipophorin-l (M, = 275,000) and apolipophorin-l I (M, = 76,000). Drosophila apolipophorin-l does not crossreact with antibodies prepared against apolipophorin-l from Manduca sexta. Key words: lipoprotein isolation, lipid composition, apolipoproteins INTRODUCTION Lipids are transported in insect hemolymph by a high-density lipoprotein called lipophorin [l-31. Lipophorin isolated from insects  contains two apoproteins, apolipophorin-I (apoLp-I* M, = 250,000) and apolipophorin-I1 (apoLp-I1 M, = 80,000); this lipophorin is composed of about 40% lipid and 60% apoproteins. The normal method for isolating insect lipophorins involves density gradient centrifugation of hemolymph . However, there are many small insects which are difficult to bleed individually in order to obtain sufficient hemolymph for isolation of lipophorins. In this paper we describe a method for isolating lipophorins by using a homogenate of the whole animal in the presence of protease inhibitors. We have chosen to use Dvosoph- Acknowledgments: We wish to thank Drs. Danny Bower and Gesina Keating for kindly providing the Drosophila. Supported by a grant from NIH (HL 39116). *Abbreviations: apolp-l = apolipophorin-I; apolp-lI = apolipophorin-ll; FITC-ConA = fluorescein isothiocyanate conjugated to concanavalin A; PAGE = polyacrylamidegel electrophoresis; SDS = sodium dodecylsulfate. Received February 5, 1988; accepted May 25,1988. Address reprint requests to Dr. Michael A. Wells, Department of Biochemistry, Biosciences West, University of Arizona, Tucson, AZ 85721. 0 1988 Alan R. Liss, Inc. 244 Fernando-Warnakulasuriya and Wells ila for these studies as a first step in using this organism as model for studying the molecular biology of lipoprotein assembly in insects. MATERIALS AND METHODS Drosophila melanogaster of the Barton strain were used in these experiments. They were reared at 29°C on the Drosophila diet provided by Carolina Biological Supply Company (Burlington, NC) . Seven to l5 grams of third-instar larvae were washed thoroughly with deionized water. Then the animals were transferred to 15 ml of ice-cold 0.1 M sodium phosphate buffer containing 0.15 M NaCl, 2 mM EDTA, 0.01% NaN3, pH = 7.0. To this solution was added 15 pl of 1 M diisopropyl phosphorofluoridate in isopropanol; 30 mg glutathione; 30 p1 of a solution containing 250 pglml of antipain, 250 pglml of leupeptin, 50 pglml of pepstatin A, and 250 pglml of chymostatin; l5 mg soybean trypsin inhibitor and 10,000 KU of aprotinin. The animals were homogenized in a Potter-Elvehjem homogenizer with the aid of a Teflon pestle. The homogenate was kept on ice for 15 min and then centrifuged in a Sorvall RC-2B centrifuge with a SS34 rotor for 30 min at 10,000 rpm and 4°C. The supernatant was carefully withdrawn with a pasteur pipette. Care was taken not to disturb the floating cake of fat. Lipoproteins were separated from this sample by using a modification of the method of Shapiro et al. . Since the Drosophila lipophorin is colorless, the lipoprotein was stained by addition of 300 pl of 10 mglml Sudan black in ethylene glycol to the sample. The stained sample was added to 8.37 g solid NaBr and the solution was made up to 20 ml with buffer. This solution was transferred to a vertical rotor centrifuge tube and overlaid with a solution of 150 mM NaC1, 0.01% EDTA, 0.01% NaN3, pH = 7.0. Centrifugation was for 16 h at 50,000 rpm and 10°C with a Beckman VTi50 rotor in a Beckman L8-70 centrifuge operating in the slow acceleration mode. The blue lipophorin band was collected as previously described . Some floating material was often observed in the tube and any of it that contaminated the lipophorin fraction was removed by passing the sample through an empty Biorad Econocolumn (i.e., a 35-pm polyethylene filter). The density of the filtrate was adjusted to 1.31 glml and the volume to 20 ml by the addition of solid NaBr and buffer and the centrifugation step was repeated. This second centrifugation was necessary to remove a slight contamination of the lipophorin by storage proteins. If necessary, the lipophorin sample was stored at 4°C without dialysis, since Drosophila lipophorin is unstable and tends to precipitate at low salt concentrations. To check the purity of the isolated lipophorin it was dialyzed against 100 mM ammonium bicarbonate, 0.001% EDTA pH = 7.9, lyophilized, and analyzed by SDS-PAGE 171. The gels were stained by using freshly prepared 0.05% Coomassie blue R-250 in 30% methanol, 20% glacial acetic acid, and 50% water. The gel was shaken gently overnight in about 300 ml of the above solution. The above-mentioned staining procedure was used since there are some apoproteins that do not stain readily in the regular stain. The protein content of lipophorin was measured according to a modification of the Lowry method  with bovine serum albumin as the standard. To determine the presence of carbohydrates, Drosophila Lipophorin 245 15 p g of lipophorin was run on a SDS gel and electroblotted onto cellulose nitrate paper and stained with FITC-ConA . Munducu sextu lipophorin was used as a standard. Lipids were analyzed as described previously [7,lO]. RESULTS AND DISCUSSION After centrifugation, lipophorin is seen as a broad band, with an average density of 1.16 glml (density limits 1.154-1.176). When lipophorin was subjected to SDS-PAGE (Fig. l),two protein bands were observed which correspond to apoLp-I and apoLp-I1 found in other insect lipophorins . Immunoblotting of the Drosophilu apoproteins with antibodies raised against M. sextu lipophorin, which contained antibodies against both apoLp-I and apoLp-11, indicated no antigenic cross-reactivity. A similar lack of crossreactivity was shown for apoLp-I from lipophorins of seven insect orders, although some species showed cross-reactivity for apoLp-I1 . The molecular weights of Drosophilu apoLp-I and apoLp-I1 are approximately 275,000 and 76,000, respectively. FITC-ConA staining shows the presence of carbohydrate chains of the high mannose type on both apoproteins (Fig. 2). The lipid composition of Drosophilu lipophorin (Table 1)shows that phospholipids are the predominant lipid with smaller amounts of acylglycerols. The relative low amount of diacylglycerol, compared to other lipophorins , may reflect the low fat content of the diet [ll].The relative high content of triacylglycerol is unusual compared to other lipophorins . We considered the possibility that additional triacylglycerol became associated with lipophorin during the homogenization procedure. Since the major source of triacylglycerol is the fat body, the following experiment was conducted. Freshly collected hemolymph from ten M. sextu larvae was divided into two aliquots. One aliquot was homogenized with the total fat body from six larvae, as described for homogenization of Drosophilu larvae, while the other was untreated. After addition of Sudan Black to both samples, lipophorin was isolated by the usual procedure. The density of the control lipophorin, and that of the lipophorin isolated from hemolymph homogenized with fat body, were identical to those previously reported 131. The ratio of diacylglycerol to phospholipid was 2.20 in the control lipophorin and 2.57 in the lipophorin isolated from hemolymph homogenized with fat body; the ratio of discylglycerol to triacylglycerol was 11 for control lipophorin and 16 for lipophorin isolated from hemolymph homogenized with fat body. Based on these data we conclude that homogenization of the fat body with lipophorin does not alter the lipid composition of the lipophorin. The fatty acid composition of the major lipids (Table 2) shows that the phospholipid is rich in 16:0, 16:1, 391, and 18:2 fatty acids. An unusual characteristic of the Drosophila acylglycerols is the high concentrations of 14:1 fatty acid, especially in the diacylglycerol fraction. The diacylglycerol fraction also has an unusually high amount of 14:O fatty acid. The triacylglycerol fraction has a high amount of 16:l. These fatty acid distributions suggest de novo synthesis of fatty acids. These results show that this whole-animal homogenization method can be used to isolate lipophorins from small animals. It should be applicable to 246 Fernando-Warnakulasuriya and Wells S+M kl S + D D 1 2 3 4 205 116 97 66 45 36 29 Fig. 1. SDS-PAGE of Drosophila lipophorin. Lane 1 (S + M) = M. sexta lipophorin plus high molecular weight standards; lane 2 (M) = M. sexta lipophorin; lane 3 (D + S) = Drosophila lipophorin plus high molecular standard; lane 4 (D) = Drosophila lipophorin. Drosophila Lipophorin D D 247 M Aw- I APO- II Fig. 2. FITC-Con A staining of lipophorins. M. sexta (lanes 1 and 2) and Drosophila lipophorins (lane 3) were separated by SDS-PAGE. After Western blotting onto nitrocellulose paper, the paper was stained with FITC-Con A and photographed under UV light. TABLE 1. Composition of DrosophiZa Lipophorin* Component Weight % Phospholipid Diacylglycerol Triacylglycerol Hydrocarbon Sterol Protein 23.1 i 5.9 7.4 1.1 5.4 3.5 0.9 & 0.5 0.7 0.1 62.5 & 5.9 ** * *Data represent average + SD (n = 3). TABLE 2. Fatty Acid Compositions of the Lipids From Drosophila Lipophorin* PLa D G ~ T G ~ 12:O 14:O 14:l 16:O Fatty acid 16:l 18:0 18:l 18:2 18:3 0.1 (0.06) 2.5 (1.3 ) 0.6 (0.02) 4.6 (0.8) 45.8 (5.9) 22.6 (3.0) 0.7 (0.3) 34.9 (3.9) 10.9 (2.4) 26.2 (0.8) 4.7 (0.8) 23.4 (1.0) 18.6 (0.9) 5.7 (1.3) 17.6 (2.5) 1.9 (0.5) 0.5 (0.1) 4.1 (2.5) 30.7 (1.4) 4.3 (0.6) 17.0 (2.2) 14.6 (2.3) 1.7 (0.7) 3.5 (2.3) 2.9 (0.4) TR' *Data (mol YO)represent average with SD given in ( ) below the average value (n = 3). aPL = phospholipid. bDG = diacylglycerol. 'TR = trace ( < 0.1 mol%). d~~ = triacylglycerol. 0.5 (0.1) 248 Fernando-Warnakulasuriyaand Wells other small animals such as ants, termites, nematodes, brine shrimp, etc., which are inconvenient to bleed. LITERATURE CITED 1. Chino H, Downer RGH, Wyatt GR, Gilbert LI: Lipophorin, a major class of lipoproteins of insect hemolymph. Insect Biochem, 21, 491 (1981). 2. Beenakkers AMTh, Van der Horst DJ, Van Marrewijk WJA: Insect lipids and lipoproteins. Prog Lipid Res, 24, 19 (1985). 3. Shapiro JP, Law JH, Wells MA: Lipid transport in insects. Annu Rev Entomol, 33, 297 (1988). 4. Ryan RO, Schmidt JO, Law JH: Chemical and immunological properties of lipophorins from seven insect orders. Arch Insect Biochem Physiol, 1, 375 (1984). 5. Shapiro JP, Keim PS, Law JH: Structural studies on lipophorin, an insect lipoprotein. J Biol Chem, 259, 3680 (1984). 6. Fernando-Warnakulasuriya GJP, Eckerson ML, Clark WA, Wells MA: Lipoprotein metabolism in suckling rats: Characterization of plasma and lymphatic lipoproteins. J Lipid Res, 24, 1626 (1983). 7. Prasad SV, Ryan RO, Law JH, Wells MA: Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexfa. J Biol Chem, 262, 558 (1986). 8. Peterson GL: Determination of total protein. Methods Enzymol, 92, 95 (1983). 9. Furlan M, Perret BA, Beck EA: Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins. Anal Biochem, 56, 361 (1979). 10. Fernando-Warnakulasuriya GJP, Staggers JE, Frost SC, Wells MA: Studies on fat digestion, absorption and transport in the suckling rat. I. Fatty acid composition and concentration of major lipid components. J Lipid Res, 22, 668 (1981). 11. Fernando-Warnakulasuriya GJP, Tscuhida K, Wells MA: Effect of dietary lipid content on lipid transport and storage during larval development of Manducu sexta. Insect Biochem, 28, 211 (1988).