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Isolation and characterization of lipophorin from Drosophila melanogaster larvae.

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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 [4] 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 [5]. 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. [5]. 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 [6]. 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 [8] 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 [9]. 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 [4].
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 [4]. 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 [3],
may reflect the low fat content of the diet [ll].The relative high content of
triacylglycerol is unusual compared to other lipophorins [3]. 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).
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isolation, melanogaster, larvae, characterization, drosophila, lipophorin
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