Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

Heterosynthetic origin of the major yolk protein,vitellin, in a nereid,Perinereis cultrifera (polychaete annelid)

• 1.1. An examination of proteins synthesized by Perinereis cultrifera oocytes incubated in vitro with [³H]leucine clearly shows that these cells are not capable of synthesizing the main yolk protein previously identified in this worm.
• 2.2. In addition, the detection of radiolabelled vitellin in oocytes after in vitro incubation of an oocyte-coelomocyte cell mixture in presence of [³H]leucine strongly suggests that the coelomocytes, free cells in the coelomic cavity, synthesize and secrete a vitellin precursor, vitellogenin, that is subsequently taken up by the oocytes.
• 3.3. Two native proteins differing in mol. wt but reacting with anti-vitellin antibodies have been identified in coelomocyte incubation medium. Also found in the coelomic fluid, they have been designated VG1 (M_r = 530,000) and VG2 (M_r = 320,000).
• 4.4. The two vitellogenins consist of a single type of polypeptide of M_r = 176,000 and are incorporated in the oocytes where they are apparently observed under a single molecular form corresponding to VG1, the highest mol. wt protein similar in size to the initial form of vitellin (VI, 530,000).
• 5.5. From these data, it seems likely that VG2 is a monomeric molecule that is taken up by the oocytes as a dimer of VG1.
• 6.6. We conclude that P. cultrifera accumulates vitellin heterosynthetically and that vitellogenin is produced by the coelomocytes. Moreover, a single polypeptide similar in size to the polypeptidic component of secreted vitellogenin has been detected in the coelomocytes.
• 7.7. Since this polypeptide has been identified previously as the single intraoocytic precursor of the four lower mol. wt products that make up the mature form of vitellin (V5), it appears that P. cultrifera exhibits for vitellogenin a processing pathway in which cleavage of the precursor occurs only after uptake by the oocyte.

     

Vitellogenin uptake and vitellin localization in insect follicles examined using monoclonal antibodies and confocal scanning microscopy

Summary

Confocal scanning immunofluorescent microscopy and monoclonal antibodies were used to examine the route of uptake of vitellogenin (VG) by vitellogenic follicles and the ooplasmic localization of vitellin (VN) in the cricket, Acheta domesticus, and the stick insect, Carausius morosus. Uptake and cytoplasmic regionalization of a non-vitellogenic sulfated protein, sp 157/85, by C. morosus oocytes were also examined. By indirect immunofluorescence VG in both species and sp 157/85 were visualized in spaces between follicle cells and in peripheral yolk spheres. One cricket VG polypeptide had a regionalized distribution in the folliclular epithelium, and VN polypeptides in both species and sp 157/85 in C. morosus had regionalized distributions within the ooplasm. Localization of sp 157/85 to the anterior pole of the oocyte appeared to be stage-specific.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Studies on vitellogenin from the tobacco hornworm,Manduca sexta

In the tobacco hornworm moth, Manduca sexta, vitellogenin (Vg) is a very high-density (1.29 g/ml) phosphoglycolipoprotein containing 13% lipids, 3% carbohydrates, and 0.6% protein-bound phosphorus. Vitellogenin (M_r～500,000) has two apoproteins designated apoVg-l (M_r 177,000 ± 3,600) and apoVg-ll (M_r45,000 ± 5,000). ApoVg-l and apoVg-II can be dissociated with 6 M guanidine HCI and separated from each other by gel permeation chromatography. Immunoblotting experiments using antibodies against the apoproteins showed that apoVg-l and apoVg-II antigens were immunologically distinct polypeptides. Antibodies against Vg reacted only with apoVg-l. Antibodies against Vg and apoVg-l reacted with Vg in double immunodiffusion experiments, whereas antibodies against apoVg-II did not. These results suggest that in the native Vg molecule, apoVg-II is positioned inside the molecule away from the aqueous environment. Only apoVg-I contained covalently bound carbohydrate as shown by fluorescein isothiocyanateconjugated concanavalin A, periodate-Schiff reagent, and in vivo labeling with ³H-Man. In vivo labeling with ³²P-inorganic phosphate and chemical determination showed that apoproteins of both Vg and vitellin contain covalently bound phosphate groups.     

Polypeptide composition and biosynthesis of the yolk proteins in the firebrat,Thermobia domestica (insecta,thysanura)

Ion-exchange chromatography of crude ovarian extracts of the primitive insect Thermobia domestica allowed the separation, in native conditions, of major and minor vitellins of molecular weights of 300,000 and 430,000, respectively. Their polypeptide subunits were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunotransfer using an antiserum prepared against major vitellin. This protein was resolved into large (M_r 166,000–212,000) and small (around M_r 50,000) polypeptides. Minor vitellin, on the other hand, exclusively contained small polypeptides that are immunologically different from those of the major vitellin. Vitellogenin polypeptides from the hemolymph of mature females exhibited electrophoretic mobilities and immunological properties similar to vitellin polypeptides. Pulse-chase experiments showed that the female fat body synthesizes radioactive and immunoprecipitable proteins, whose polypeptide pattern is close to that of the major vitellogenin. However, part of the primary vitellogenic polypeptides, at M_r 210,000 and 212,000, is rapidly processed to M_r 176,000 and 182,000 subunits. These two polypeptides, as well as the precursors, enter into the composition of the major hemolymph vitellogenin. Finally, processing of the still uncleaved 210,000–212,000 polypeptides takes place in the ovary, which performs the same step of vitellogenin maturation as the fat body.     

Postendocytic vitellin processing in ovarian follicles of the stick insect Carausius morosus (Br.)

Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A₁, A₂, and A₃) and 2 (B₁ and B₂) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV follicles, Vit A resolved into 4 distinct vitellin polypeptides (A₀, A₁, A₂ and A₃). Using a panel of monoclonal antibodies, polypeptide A₀ proved to be immunologically related to polypeptide A₂. In follicles about to begin choriongenesis, polypeptide A₃ was gradually replaced by a lower M_r polypeptide. Over the same time period, polypeptide B₁ changed in charge, but not in M_r. To confirm the existence of a polypeptide processing in C. morosus, ovarian follicles of different developmental stages were exposed in vivo to [³⁵S]-methionine from 6 to 72 h. Data showed that A₀ and B₁ were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B₂ and A₃ were also labelled to some extent. With progressively longer exposures, polypeptides A₁ and A₂ also became labelled. In vivo exposure to [³H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of ovarian follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.     

Purification,characterization, and synthesis of vitellin from the cabbage butterfly Pieris rapae L.

Vitellin from the cabbage butterfly Pieris rapae L. was purified and characterized by electrophoresis. Vitellin from P. rapae is a phosphorylated glycolipoprotein of 380,000 ± 10,000 molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis. Two subunits with an M_r of 150,000 and 40,000 were obtained from vitellin. The native molecule is thought to be a tetramer composed of two molecules of each of these subunits. The isoelectric point, as determined by isoelectric focusing on polyacrylamide gels, is 6.10. Vitellin and vitellogenin were indistinguishable by immunological methods such as double diffusion and tandem-crossed immunoelectrophoresis. Vitellogenin from the hemolymph and vitellin from the ovary were quantified by rocket immunoelectrophoresis. Vitellogenin and vitellin were first detected in 6-day-old pupae, and their levels increased continuously during ovarian development. Vitellogenin synthesis by the fat body in 4-day-old female pupae could be induced by juvenile hormone I.     

Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake

Two classes of vitellogenin binding sites with K_d-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (K_d=16.6 nM), whereas loweraffinity binding sites (K_d=200 nM) were found in the oocyte membrane fraction. The concentration of Ca²⁺ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca²⁺ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca²⁺. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.     

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout,Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (¹²⁵iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K _d=8.2·10^-9 mol·1^-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B _max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K _d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Selective endocytosis,in vitro,by ovarian follicles from Hyalophora cecropia

Ovarian follicles of Hyalophora cecropia, incubated in vitro with isolated and radiolabelled hemolymph and yolk proteins, provided a satisfactory model of in situ vitellogenesis. Uptake of proteins was specific. The follicles accumulated vitellogenin and microvitellin at constant rates for 6 hr, depositing them in the protein yolk spheres of the oocyte. Uptake of these two proteins was saturable by high concentrations of homologous protein and inhibited by p-dinitrophenol. In contrast, two other abundant hemolymph proteins, arylphorin and flavoprotein, were taken up at lower rates, and become concentrated primarily in the basement lamina of the follicle. Their accumulation was not saturable and not inhibited by p-dinitrophenol. The two yolk precursors were accumulated only by follicles at stages known to be vitellogenic, and the rates of uptake were shown to approximate the rates of accumulation of these proteins in situ. The uptake of vitellogenin, but not microvitellin, was enhanced 2- to 3-fold by hemolymph ultrafiltrates. Vitellin from mature eggs was not distinguishable from vitellogenin by the endocytotic apparatus. Finally, endocytotic uptake was not affected by inhibition of protein synthesis. This finding supports the concept of membrane and receptor recycling in yolk formation, and argues against an essential role of the follicle cell product paravitellogenin in the mechanism of hemolymph protein uptake.     

Immunolocalization of a proton V-ATPase in ovarian follicles of the tobacco hornworm Manduca sexta

A monoclonal antibody, directed against an H⁺ translocating V-ATPase of the midgut of Manduca sexta, has been used for immunolocalization studies in ovarian follicles and testes of Manduca sexta. In testes, no distinct staining above background levels was observed. In vitellogenic follicles, V-ATPase immunoreactivity first appears in the cytoplasm of the trophocytes and then in the oocyte, but by far the strongest reaction is present in the region of the oolemma during endocytosis. All types of follicle cells surrounding both the oocyte and the trophocyte compartments show a distinct positive reaction. In the cylindrical follicle cells surrounding the oocyte, the immunoreactivity is clearly restricted to the basal part. Our results suggest an important role for V-ATPase in vitellogenin uptake in Manduca, similar to that suggested on electro-physiological grounds in Hyalophora cecropia. © 1995 Wiley-Liss, Inc.     

Studies on the carbohydrate moiety of vitellogenin from the tobacco hornworm,Manduca sexta

Vitellogenin can be isolated in large quantities from the hemolymph of the tobacco hornworm, Manduca sexta by a combination of KBr density gradient ultracentrifugation, gel permeation and cation exchange chromatography. Glycopeptides generated by exhaustive pronase digestion of vitellogenin were separated by gel permeation chromatography on a Bio-Gel P-6 column. The major glycopeptide was shown to bind to concanavalin A-Sepharose but not to lentil lectin-Sepharose and was sensitive to treatment with endo-β-N-acetylglucosaminidase H. Analysis of the glycopeptide by high field proton NMR spectroscopy revealed that the primary structure is of the high mannose class containing nine mannose and two N-acetylglucosamine units. These results suggested that N-glycosylation of insect proteins, as in mammals, yeasts and plants, involves the en bloc transfer of an oligosaccharide containing the unit (Glu)₃(Man)₉(GlcNAc)₂ from a lipid intermediate to an asparagine residue followed by removal of glucose residues. Processing to more complex structures does not seem to occur in M. sexta vitellogenin. In vitro uptake with isolated M. sexta follicles showed that deglycosylation had no significant effect on uptake of ¹²⁵I-labeled vitellogenin.     

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M_r = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 ± 20 μg/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH₂-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses. © 1994 Wiley-Liss, Inc.     

Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [³²P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with ³²P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term ³²P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with ³²P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro³²P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify ³²P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.     

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and ¹²⁵I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[¹²⁵I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of ¹²⁵I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of M_r 120 000 was internalized by macrophages somewhat more rapidly than copolymer of M_r 46 000, but was excluded from the yolk sac.     

Chemical and immunological properties of lipophorins from seven insect orders

The major insect hemolymph lipoprotein, lipophorin, was isolated from adults of eight insect species representing seven insect orders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to compare their respective apoprotein components. In all species examined lipophorin was composed of at least two apoproteins, apolipophorin I (M_r ～ 250,000) and apolipophorin II (M_r ～ 78,000), and two species had a third apoprotein, apolipophorin III (M_r ～ 17,000). The density of each isolated lipophorin was determined from the refractive index of KBr following density gradient centrifugation. Immunoblotting with anti-larval Manduca sexta apolipophorin I and II of the apoproteins separated by SDS-PACE indicated cross reactivity between anti-M sexta apoLp-ll and apoLp-ll in all species tested. Anti-M sexta apoLp-l exhibited no cross reactivity for any species tested. Fluorescent lectin staining of the apoproteins separated on SDS-PAGE gels revealed the presence of covalently bound carbohydrate residues.