Archives of Insect Biochemistry and Physiology 19:133-144 (1992) Transfer of Phospholipids From Fat Body to lipophorin in Rhodnius prolixus Georgia C o d a Atella, Katia Calp Gondim, and Hatisaburo Masuda Departamento de Bioqufmica Mkdica, lnstituto de Citncias Biomiddicas, Universidade Federal do Rio de Janeiro, RJ, Brasil 32P-Labeledfat bodies (32P-fatbodies) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin i s a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of preLlabeled 32P-lipophorinby the tissue. Analysis of the 32P-phospholipidstransferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximalat about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32Pphospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis. Key words: lipid carrying protein, lipid, lipoprotein Acknowledgments: We wish to express our gratitude to Dr. Martha M. Sorenson for a critical reading of the manuscript; to Rosane O.M.M. Costa, Jose de S. Lima, Junior and Jose F. de Sousa Net0 for excellent technical assistance. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Financiadora de Estudos e Projetos (Finep), Fundaqao de Amparo 21 Pesquisa do Rio de Janeiro (Faperif and Conselho de Ensino para Graduados (CEPC) of the Universidade Federal do Rio de Janeiro. Received July18,1991 ; accepted November 4,1991. Address reprint requests to Katia Calp Condim, Departamentode Bioquimica Medica, lnstituto de [email protected], Universidade Federal do Rio de Janiero, RJ, Brasil, 21910. 0 1992 Wiley-Liss, Inc. 134 Atella et al. INTRODUCTION In insects lipids are transported by lipophorin, a major hemolymph lipoprotein which contains a large amount of lipids [l-31. Diacylglycerols, cholesterol, hydrocarbons, and phospholipids are the major lipids associated with lipophorin [4-71. Lipophorin takes up diacylglycerols from the fat body and transports them to flight muscles and oocytes [5,8-lo]. Lipophorin also transports hydrocarbons from the oenocytes, where they are synthesized, to the cuticle and takes up cholesterol from the digestive tract [6,11]. The transport of diacylglycerols is the most studied lipid transport system in insects. In insect species that use lipids to obtain energy for sustained flight, diacylglycerolsare loaded onto high density lipophorin particles at the fat body. As a consequence, the ratio of lipids to protein increases and as lipophorin becomes less dense, it is termed low density lipophorin [12J.These changes are mediated by adipokinetichormone [9,13-151. The diacylglycerols from LDLp* are transferred to the flight muscles to be used as fuel for flight [16,17]. After unloading of the diacylglycerolsat the muscles the lipophorin particles become denser. This HDLp can be reloaded with more diacylglycerols at the fat body and recycled . Other physiological situations, such as oogenesis, also require large amounts of lipids. The developing oocytes accumulate lipids in the form of droplets; Wiemerslage  and Chino et al.  proposed that diacylglycerols are transported by lipophorin to the oocytes. This was confirmed by Kawooya and Law [lo] in Manducu sextu. Gondim et al.  showed that during oogenesis, in Rhodnius prolixus phospholipids can be transferred from lipophorin particles to the growing oocytes and that the lipophorin apoproteins are not accumulated in the oocytes, suggesting that lipophorin may function as a shuttle for phospholipids as well as for diacylglycerols. The question of whether phospholipids are transportable has been debated but is still not clear. Although Thomas and Gilbert  have shown the release of phospholipids from fat body to the hemolymph in Hyulopkaru cecropia, Katagiri and Chino  could not confirm their results. On the basis of lipophorin structural studies, Katagiri  proposed that phospholipids are components of the lipophorin particle but are not transferred to tissues. To answer the question of whether Iipophorin particles can be considered a reusable shuttle for phospholipids, it is necessary to show that the particles can be reloaded with more phospholipids. In this report we demonstrate that lipophorin particles can be loaded with phospholipids at the fat body, thus supporting the concept that lipophorin can in fact be considered a reusable shuttle for phospholipids. MATERIALS AND METHODS Insects Insects were taken from a colony of Rkodnius prolixus maintained at 28°C and 70-80% relative humidity. The experimental insects were adult, mated females fed on rabbit blood at 2-week intervals. *Abbreviations used: HDLp = high density lipophorin; LDLp = low density lipophorin; LTP = lipid transfer particle; PBS = phosphate-buffered saline; SDS = sodium dodecyl sulfate. Transfer of Phospholipids in Rhodnius 135 32PiPurification Carrier-free 32Pipurchased from Comissao Nacional de Energia Nuclear (Siio Paulo, Brasil) was purified by extraction of the phosphomolybdate complex . Lipophorin Purification Two to 5 days after a blood meal, hemolymph was collected in the presence of phenylthiourea (30-130 Fg/pl), 5 mM EDTA, and a mixture of protease inhibitors prepared in 0.15 M NaC1, with final concentrations of 0.05 mg/ml soybean trypsin inhibitor, leupeptin, and antipain, and 1mM benzamidine. The collected hemolymph was centrifuged at room temperature for 5 min at 13,0009 and lipophorin was purified from the supernatant as described previously . The supernatant was diluted to 5 ml with 5 mM EDTA and PBS (10 mM phosphate, 0.15 M NaCI, pH 7.4) and 1.25 g KBr was added. This material was centrifuged at 159,0009 in a Beckman (Santa Clara, CA)Type 50 rotor at 4°C for 20 h and lipophorin was collected from the top of the KBr gradient. The purified lipophorin was extensively dialysed against PBS and 5 mM EDIA and then against PBS, and was stored under liquid nitrogen until use. The degree of purification was monitored by SDS-PAGE and the protein concentration was estimated according to Lowry et al. , using bovine serum albumin as standard. Polyacrylamide Gel Electrophoresis Polyacrylamide slab gels were run both under denaturing conditions (with SDS, ) and under nondenaturing conditions . For radioactive samples, the gels were stained, photographed, dried, and autoradiographed. Preparation of 32P-LabeledFat Body (32P-Fat Body) Adult females were fed on blood enriched with 32Pi (lo9 c p d m l of blood) , using a special feeder described by Garcia et al. . Two days after a blood meal, the insects were carefully dissected and the 32P-fatbody was left untouched and associated with the abdominal cuticle. Transfer of Radioactivity From 32P-Fat Bodies to Lipophorin The 32P-fatbodies were extensively washed in an excess of Rhodnius Ringer [31J and then in 0.15 M NaC1, to remove contaminating radioactive hemolymph. Twenty microliters of culture medium (no. 199, Sigma, St. Louis, MO) containing nonradioactive purified lipophorin was added to the washed 32P-fatbodies. Unless otherwise stated, the incubationswere performed at 28°C. At the desired time, culture medium was taken (10 PI), diluted to 100 pl with 0.15 M NaCl, and centrifuged at 13,OOOg for 10 min. The supernatants were separately applied to Sephadex G-50 centrifuge columns  previously equilibrated with PBS to separate the proteins from the small phosphorylated molecules. The eluted material was analysed by PAGE and autoradiography or by scintillation counting. Controls were done by incubating 32P-fatbodies in culture medium without lipophorin. In the experiments performed in the presence of metabolic inhibitors the 32P-fatbodies were preineubated for 1 h at 28°C in the culture medium containing the inhibitors but in the absence of lipophorin. After the preincubation, nonradioactive lipophorin was added and the incubation was continued for 30 min. The samples were treated as described above. 136 Atella et al. Determination of the Amount of Phospholipids Transferred From Fat Body to Lipophorin Nonradioactive fat bodies prepared in the same way described for radioactive fat bodies were incubated for 2 h in the presence of 17.0 mg/ml of nonradioactive lipophorin. After incubation of 8 fat bodies, the culture media were pooled and centrifuged for 10 min at 13,OOOg and the lipophorin was repurified from supernatant in a KBr ultracentrifugation gradient as described before. After dialysis against PBS, the repurified lipophorin was subjected to lipid extraction  as described elsewhere , and the amount of phospholipids was determined by measuring the phosphate content according to a modified procedure of Bartlett  as described by Kates . Phospholipid Analysis After incubation of 32P-fatbodies with purified lipophorin, the culture media were pooled and centrifuged for 10 min at 13,OOOg and the supernatant was applied to a KBr gradient as described above for the repurification of lipophorin (now radioactive). The purified 32P-lipophorinwas separated from KBr by dialysis and was then subjected to lipid extraction . The radioactivity of the lipid moiety was determined separately by scintillation counting. The 32P-phospholipidsextracted from the 32P-lipophorinwere analysed by two-dimensional thin-layer chromatography, as described by Yavin and Zutra  with slight modifications . The plates were stained with iodine and autoradiographed. RESULTS When 32P-fatbodies were incubated in culture medium in the presence of nonradioactive purified lipophorin, a significant amount of radioactivity was secreted to the medium, where it was associated with lipophorin (Fig. 1, lane 4). Without addition of lipophorin no radioactivity appeared in the medium, showing that there was no release of 32P-lipophorinby the 32P-fatbody during the incubation period (Fig. 1, lane 2). To be sure that the 32P-phospholipids were transferred to lipophorin from the fat body and not from the cuticle, lipophorin was incubated in the abdominal cuticle after removal of the 32P-fat bod No transfer of radioactivity to lipophorin was observed (Fig. 1, lane 3). No 1;P-vitellogenin was detected in the medium under all conditions used, probably because of the short incubation period. To ascertain whether the radioactivity transferred from the 32P-fatbodies to nonradioactive lipophorin was associated with phospholipids, the 32P-lipophorin obtained after incubation was purified and subjected to lipid extraction, as described in Materials and Methods. All the radioactivity was found in the lipid fraction. The phospholipids were analysed by thin-layer chromatography followed by autoradiography (Fig. 2); phosphatidylethanolamineand phosphatidylcholinewere the major phospholipids found. Phosphatidylserine, phosphatidylinositol, and sphingomyelin were also observed. The time-course of the transfer of "P-phospholipids from 32P-fatbodies to lipophorin is shown in Figure 3. After a rapid initial phase, the phospholipids were transferred to lipophorin at a constant rate for at least 45 min. In the Transfer of Phospholipidsin Rhodnius 137 Fig. 1. PACE analysis under nondenaturing conditions (5-10% polyacryiamide gradient) of the culture medium obtained after incubation of 32P-fatbodies with nonradioactive lipophorin. A: Coomassie blue stained gel. B: Autoradiography of the gel. lane 1. Hemolymph from a female 2 days after feeding 32Piin a blood meal (control to show the positions of (LP) lipophorin and (VG) vitellogenin). lane 2. Culture medium from incubation of 32P-fatbody without purified lipophorin. Lane 3. Culture medium containing nonradioactive lipophorin incubated in abdominal cuticle after removal of 32P-fatbody. lane 4. Culture medium from incubation of 32P-fat body with nonradioactive lipophorin. The incubations were for 15 min at 28°C and the lipophorin concentration was 12 mg/ml. Other experimental conditions were as indicated in Materials and Methods. absence of lipophorin (lower curve, Fig. 3), a negligible amount of radioactivity appeared in the medium. The transfer of phospholipids to lipophorin was abolished at 0°C and reduced in the presence of metabolic inhibitors such as sodium azide or sodium fluoride (Fig. 4). The rate of 3ZP-phospholipidtransfer to lipophorin increased with increasing concentrations of lipophorin in the medium, with a tendency to saturation (Fig. 5). To show whether the plateau observed at the highest lipophorin concentrations was due to a real saturation and not due to depletion of 32P-phospholipidsof fat bodies, a control was performed by incubating 32P-fat bodies at the highest lipophorin concentration (40 mg/ml) for a longer period of time. The transfer of phospholipids was twice that observed after 15 min of incubation indicating that the plateau in Figure 5 reflects saturation and not depletion. It seems that the transfer of phospholipids from fat body is, in fact, mediated by a receptor for lipophorin. The concentration of lipophorin 138 Atella et al. Fig. 2. Autoradiography of a two-dimensional thin-layer chromatogram of phospholipids extracted from 32P-lipophorinobtained by incubation of nonradioactive lipophorin with 32P-fat bodies. Nonradioactive lipophorin (6 mg/ml) incubated for 60 min with 32P-fatbodies was repurified and the phospholipids were extracted. Phosphatidylethanolarnine (PE); phosphatidylcholine (PC); phosphatidylserine (PS); sphingomyelin (SM);phosphatidylinositol (PI).The first and the second dimensions are indicated in the figure. The experimental conditions were as described in Materials and Methods. required to produce the half maximal rate of phospholipid transfer was about 8 mg/ml. The amounts of phospholipids associated with lipophorin before and after the incubation with fat bodies were determined by their phosphate contents and compared (Table 1) to show that the incorporation of radioactivity by lipophorin was due to a net gain of phospholipids and not to an exchange. The net gain of phospholipids was 8.54 nmolhnsect in 2 h incubation, which corresponds to an increase of 6.69%in the lipophorin phospholipid content. Analysis of the rate of 32P-phospholipidsecretion by 32P-fatbodies to lipophorin showed that during the days following the blood meal the rate increased up to day 10 and then decreased (Fig. 6). Transfer of Phospholipids in Rhodnius 10 0 20 30 139 40 TIME (min) Fig. 3. Time-course of 32P-phospholipidtransfer from 32P-fatbodies to lipophorin in vitro. "P-fat bodies were incubated without (0) or with ( 0 ) purified lipophorin (5 r n g h l ) and at different times an aliquot (10PI) of the medium was taken, the32P-lipophorinwas separated from the small molecules and the amount of radioactivity associated with lipophorin was estimated by scintillation counting. Other experimental conditions were as described in Materials and Methods. The vertical bars represent the SE for 4 determinations. A C D E Fig. 4. Effects of metabolic inhibitors and low temperature on 32P-phospholipidtransfer from '*P-fat bodies to lipophorin. 32P-fatbodies were incubated for30 min at28"C in a culture medium containing 8 mg/ml nonradioactivelipophorin and no added inhibitor (A); lipophorin plus 10 rnM NaN3(6);lipophorin plus 10 mM NaF (C);no lipophorin (D); or lipophorin with no added inhibitor but at 0°C (E). After the incubation the radioactivity transferred to lipophorin was estimated as described in Materials and Methods. The vertical bars represent the SE for 4 determinations. 140 Atella et al. 0 10 20 30 40 LIPOPHORIN CONCENTRATION (mg/ml) Fig. 5. Effect of lipophorin concentration on the transfer of 32P-phospholipids.32P-Fatbodies were incubated with different lipophorinconcentrations for 15 min ( 0 )or30 min (A).The radioactivity transferred to lipophorin was determined by scintillation counting, as described in Materials and Methods. The vertical bars represent the SE for 4 determinations. DISCUSSION We have shown previously that lipophorin transfers phospholipids to growing oocytes and that the lipophorin apoproteins are not accumulated by the ovary . Here we present evidence to support the concept that lipophorin is a reusable shuttle for phospholipids by showing that lipophorin can be loaded with phospholipids (by showing that lipophorin can be loaded with phospholipids) at the fat body. Prelabeled 32P-fatbodies incubated with nonradioactive purified lipophorin released radioactivity to the lipoprotein (Fig. 1).The presence of two lipophorin bands was described earlier , but their biological significance is still unknown. The radioactivity bound to lipophorin particles TABLE 1. Transfer of Phospholipids From Fat Body to Lipophorint Control incubated without fat body After incubation with fat body Phospholipids transferred Phospholipid content in lipophorin particles (nmol) Percentage 128.0 f 3.2* 136.5 k 3.3" 8.5 k 1.2 100 106.7 6.7 tThe amounts of phospholipids associated with lipophorin particles were measured as described in Materials and Methods. The values represent the content of phospholipids associated with 340 kg of lipophorin protein, which was the total amount of lipophorin used to challenge the fat body of one insect. The incubation period was 2 h. Data are means SE for 4 determinations. "The difference between the 2 groups is significant for P < .05. Transfer of Phospholipids in Rhodnius 141 days after feeding Fig. 6. Rate of phospholipid transfer from 32P-fatbody to lipophorin during the days after a blood meal. Insects were fed on day zero with blood enriched with 32Pi, At different days following the meal the "P-fat bodies were isolated in the abdominal cuticle and incubated with 3.5 mg/rnl nonradioactive lipophorin for 30 min at 28°C. Data are corrected for 32P decay. The vertical bars represent the SE for 4 determinations. was associated solely with the phospholipid moieties (Fig. 2). As it has been observed for diacylglycerols,hydrocarbons, and cholesterol [5,6,9,11],lipophorin can be loaded with phospholipids (Table 1). This observation brings about the conclusion that phospholipids are not only part of the lipoprotein vehicle itself, but they are also a transportable lipid, confirming the initial results of Thomas and Gilbert , who showed the release of phospholipids from fat bodies incubated in the presence of hemolymph in Hyalophora cecropia. It is not clear why Katagiri and Chino  did not observe the transfer of phospholipids to lipophorin in vitro in Philosamia cynthia or Hyalophora cecropia. The transfer of 32P-phospholipidsto lipophotin was linear with time (Fig. 3). The control, in the absence of lipophorin in the medium, showed that even after 45 min of incubation the fat bodies did not release a significant amount of radioactivity. This is a good indication that the tissues were still healthy under our experimental conditions because no leakage was observed. The process of phospholipid loading of lipophorin at the fat body was inhibited by low temperature and by metabolic inhibitors (Fig. 4).Thus, the loading process is dependent on active metabolism as it is for diacylglycerols and hydrocarbons [5,11]. The transfer of diacylglycerols from lipophorin to the flight muscles in Locusta migratoriu  and to the fat body in Munduca sextn  is mediated by lipophorin receptors found in these tissues, and the transfer of phospholipids from the fat body to lipophorin of Rhodnius is also a saturable process (Fig. 5). These data suggest that receptors are necessary for lipophorin to load and unload different lipids. The lipophorin titer in Rhodnius female hemolymph was shown to be about 40 mg/ml and the lipophorin concentration required 142 Atella et al. to produce half-maximal transfer of 32P-phospholipidfrom 32P-fatbodies was estimated to be about 8 mg/ml (Fig. 5). Considering the observed constant and the high level of circulating lipophorin concentration in the hemolymph, it can be concluded that the loading system at the fat body works at maximal velocity. The fat body capacity for loading phospholipids onto lipophorin particles increases up to day 10 after a blood meal (Fig. 6 ) coinciding with the period when most of the eggs are produced (unpublished observation) and then decreases in parallel with the process of oogenesis. The variable loading rates in the days following a blood meal cannot be attributed to changes in the radioactivity associated with fat body phospholipids which remained constant throughout the experiment (data not shown). In Manduca sextu and Locustu migrutoriu a very high density lipoprotein in the hemolymph mediates the transfer of lipids between lipophorin of different densities in vitro. This LTP [39,40] has been shown to be necessary for the transfer of diacylglycerols from the fat body to lipophorin in Manducu . Some LTP seems to be associated with the fat body, even after washing it. In this work the transfer of phospholipids to lipophorin was obtained in the presence of purified lipophorin without addition of LTP, but the existence of LTP bound to the fat body is possible. Probably, the site of synthesis of lipophorin in Xhodnius prolixus is the fat body, as it is in Munducu sextu larvae (421 and in Locusfa rnigratoria . In our experimental conditions we did not observe lipophorin secretion by the 32P-fat bodies possibly because of the very short incubation periods employed and the low turnover rate of lipophorin apoproteins, which has been measured in Locustu migrutoriu . Differing from Katagiri's  conclusion that phospholipids are only structural components of the carrier system, this study demonstrates that they are also a transportable lipid. These data taken together with the demonstration that phospholipids are in fact transferred from lipophorin to the oocytes  support strongly the idea that lipophorin acts as a reusable shuttle for phospholipids. LITERATURE CITED 1. Chino H, Downer RGH, Wyatt GR, Gilbert LI: Lipophorins, a major class of lipoproteins of insect haemolymph. Insect Biochem 11,491 (1981). 2. Chino H: Lipid transport: Biochemistry of hemolymph lipophorin. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA,Gilbert LI, eds. Pergamon Press, Oxford, vol10, pp 115-134 (1985). 3. Shapiro JP, Law JH, Wells MA: Lipid transport in insects. Annu Rev Entomol 33, 297 (1988). 4. Thomas KK, Gilbert LI: Isolationand characterization of the hemolymph lipoproteins of the American silkmoth, Hyulophoru cecropia. Arch Biochem Biophys 127,512 (1968). 5. Chino H, Murakami S, Harashima K Diglyceride-carryinglipoproteins in insects hemolymph. Isolation, purification, and properties. Biochim Biophys Acta 176,1(1969). Transfer of Phospholipidsin Rhodnius 143 6. Chino H, Gilbert LI: The uptake and transport of cholesterol by haemolymph lipoproteins. Insect Biochem 1,337 (1971). 7. Peled Y, Tietz A: Isolation and properties of a lipoprotein from the haemolymph of the locust, Locusta mzgrutoriu. Insect Biochem 5,61(1975). 8. Van der Horst DJ, Houben NMD, Beenakkers AMT: Dynamics of energy substrates in the haemolymph of Locusta migratoria during flight. J Insect Physiol26,441 (1980). 9. Beenakkers AMT, Van der Horst DJ, Van Marravijk WJA Metabolism during locust flight. Comp Biochem Physiol [BI69,315 (1981). 10. Kawooya JK, Law JH: Role of lipophorin in lipid transport to the insect egg. J Biol Chem 263,8748 (1988). 11. Katase H, Chino H: Transport of hydrocarbons by the lipophorin of insect hemolymph. Biochim Biophys Acta 720,341 (1982). 12. Beenakkers AMT, Chino H, Law JH: Lipophorin nomenclature. Insect Biochem 28,1(1988). 13. Ryan RO, Law JH: Metamorphosis of a protein. BioEssays 1,250 (1984). 14. Van Heusden MC, Van der Horst DJ, BeenakkersM In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Lacustu migrutoria. J Insect Physiol30,685 (1984). 15. Chino H, Kiyomoto Y, Takahashi K: In vitro study of the action of adipokinetic hormone in locusts. J Lipid Res 30,571 (1989). 16. Van der Horst DJ, Van Doorn JM, de Keijzer AN, Beenakkers AMT: Interconversions of diacylglycerol-transporting lipoproteins in the haemolymph of Locusta rnigratorza. Insect Biochem 17,717 (1981). 17. Wheeler CH, Van der Horst DJ, Beenakkers AMT: Lipolytic activity in the flight muscles of Locusta migrutoria measured with haemolymph lipoproteins as substrates. Insect Biochem 14,261 (1984). 18. Van Heusden MC, Van der Horst DJ, Voshol J, Beenakkers AMT: The recycling of protein components of the flight-specific lipophorin in Locusta rnigratorza. insect Biochem 2 7, 771 (1987). 19. Wiemerslage LJ: Lipid droplet formation during vitellogenesis in the cecropia moth. J Insect Physiol22,41(1976). 20. Chino H, Downer RGH, Takahashi K: The role of diacylglycerol-carrying lipoprotein I in lipid transport during insect vitellogenesis. Biochim Biophys Acta 487,508 (1977). 21. Gondim KC, Oliveira PL, Masuda H: Lipophorin and oogenesis in Rhodnius prolixus: Transfer of phospholipids. J Insect Physiol35,19 (1989). 22. Thomas KK, Gilbert LI: In vitro studies on the release and transport of phospholipids. J Insect Physioll3,963 (1967). 23. Katagiri C, Chino H: Studies on phospholipid transport by haernolymph lipoproteins. Insect Biochem3,429 (1973). 24. Katagiri C: Structure of lipophorin in insect blood: Location of phospholipid. Biochim Biophys Acta 834,139 (1985). 144 Atella et al. 25. de Meis L: Pyrophosphate of high and low energy: Contribution of pH, CaZ+,Mg’ and water to the free energy of hydrolysis. J Biol Chem 259,6090 (1984). 26. Gondim KC, Oliveira PL, Coelho HSL, Masuda H: Lipophorin from Rhodnius prolixus: Purification and partial characterization. Insect Biochem 19,153 (1989). 27. Lowry OH, Rosebrough NJ, Farr AR, Randall RJ: Protein measurements with the Folin phenol reagent. J Biol Chem 193,265 (1951). 28. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 (1970). 29. Davis BJ: Disc electrophoresis-11. NY Acad Sci 121,404 (1964). Methods and application to human serum proteins. Ann 30. Garcia ES, Macarine JD, Garcia MLM, Ubatuba FB: Alimentasiio do Rhodnius prolixus no laboratbrio. An Acad Brasil [email protected],539 (1975). 31. Maddrell SHP: Secretion by the malpighian tubules of Rhodnius. The movements of ions and water. J Exp Biol. 51,71(1969). 32. Penefsky H: Reversible binding of Pi by beef heart mitochondria1adenosine triphosphatase. J Biol Chem 252,2891 (1977). 33. Bligh EG and Dyer WJ: A rapid method of total lipid extractionand purification. Can J Biochem Physiol37,911(1959). 34. Bartlett GR: Phosphorous assay in column chromatography. J Biol Chem 234,466 (1959). 35. Kates M: In: Techniques of Lipidology. Isolation, Analysis and Identification of Lipids. Work TS, Work E, eds. North-Holland, Amsterdam, pp 114-115, (1972). 36. Yavin E, Zutra A: Separation and analysis of 3’P-labelled phospholipids by a simple and rapid thin-layer chromatographic procedure and its application to cultured neuroblastoma cells. Anal Biochem 80,430 (1977). 37. Hayakawa H: Characterization of lipophorin receptor in locust flight muscles. Biochim Biophys Acta 919,58 (1987). 38. Tsuchida K, Wells MA: Isolation and characterization of a lipoprotein receptor from fat body of an insect, Munducu sextu. J Biol Chem 265, 5761 (1990). 39. Ryan RO, Sarvamangala VP, Henriksen EJ, Wells MA, Law JH: Lipoprotein interconversions in an insect, Manducu sextu. Evidence for a lipid transfer factor in the hemolymph. J Biol Chem261,563 (1986). 40. Ryan RO, Haunerland NH, Bowers WS, Law JH: Insect lipid transfer particle catalyses diacylglycerolexchange between high-density and very-high-density lipoproteins. Biochim Biophys Acta 962,143 (1988). 41. Van Heusden MC, Law JH: An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein. J Biol Chem 264,17287 (1989). 42. Prasad SV, Fernando-Wamakulasuriya JP,Sumida M, Law JH, Wells MA: Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Munduca sextu. J Biol Chem 261,17174 (1986). 43. Gellissen G , Wyatt GR: Production of lipophorin in the fat body of adult Locustu migrutoriu: comparison with vitellogenin. Can J Biochem 59,648 (1981). 4.4. Downer RGH, Chino H: Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph. Insect Biochem 15,627 (1985).