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Biomedical subjects

R O Ryan

Publications and source records attributed to R O Ryan.

At least 73 records · Page 4Linked to original sources
PubMed1991

Displacement of apolipophorin III from the surface of low density lipophorin by human apolipoprotein A-I.

A hybrid low density lipophorin particle (LDLp) was prepared by incubation with human apolipoprotein (apo) A-I in vitro. ApoA-I associated with LDLp in a concentration dependent, saturable manner which was accompanied by dissociation of apolipophorin III (apoLp-III). The apoA-I hybrid LDLp had the same lipid composition, density and morphology as native LDLp indicating that displacement of apoLp-III by apoA-I did not affect its structural properties. The molar ratio of apoLp-I:apoLp-II:apoLp-III was maximally reduced from 1:1:16 to 1:1:2 in native versus hybrid LDLp with the latter particle binding 7 molecules of apoA-I. The inability of apoA-I to displace the remaining 2 apoLp-III supports the concept that these apoLp-III molecules are not equivalent to the other fourteen. Native and hybrid LDLp particles were both metabolized to high density lipophorin in vivo. The displacement reaction represents a novel method for the production of apolipoprotein hybrids of LDLp and the results indicate that apoA-I has an inherently higher affinity for lipid surfaces than apoLp-III.

Animals↗
PubMed1991

Role of lipid transfer particle in transformation of lipophorin in insect oocytes.

Oocyte development in Manduca sexta involves the deposition of large amounts of lipoprotein-derived lipid. One source of this lipid is hemolymph high-density lipophorin-adult (HDLp-A) which, upon entry into the oocyte, is transformed into a lipid and apolipophorin III deficient product particle, egg very-high- density lipophorin (VHDLp-E; density = 1.24 g/ml; Kawooya et al. (1988) J. Biol. Chem. 263, 8740-8747). An in vitro model of this transformation has been established using human low-density lipoprotein (LDL) as acceptor of HDLp-A associated lipid in a reaction catalyzed by isolated M. sexta hemolymph lipid transfer particle (LTP). Facilitated vectorial net transfer of lipid from HDLp-A to LDL resulted in formation of a very-high-density lipophorin (VHDLp) product with a density and apolipoprotein content similar to that of VHDLp-E. Lipid was found to comprise 25% of the VHDLp particle mass, whereas over 50% of HDLp-A mass is lipid. Based on these observations it was hypothesized that a lipid transfer factor may be present in M. sexta oocytes and function in the transformation of HDLp-A to VHDLp-E in vivo. Transfer activity was present in the buffer soluble fraction of oocyte homogenates and purification of the active material revealed a catalyst with electrophoretic and immunological properties identical to hemolymph LTP. Incubation of 125I-HDLp-A with an M. sexta oocyte homogenate resulted in transformation of the radiolabeled lipoprotein to a density corresponding to that of VHDLp-E. When the incubation media was preincubated with anti-LTP IgG this conversion was inhibited to a large extent. Inhibition was relieved, however, by addition of exogenous LTP. The results provide the first demonstration of M. sexta LTP in a tissue other than hemolymph and support the concept that LTP-catalyzed lipid transfer plays an integral role in the conversion of HDLp-A to VHDLp-E in vivo.

Animals↗
PubMed1991

Lipid transfer particle-catalyzed transfer of lipoprotein-associated diacylglycerol and long chain aliphatic hydrocarbons.

Following injection of [1-14C]acetate into the sphinx moth, Manduca sexta, radiolabel was incorporated into lipid components of the major hemolymph lipoprotein, adult high density lipophorin (HDLp-A). Analysis of the labeled lipids by thin layer chromatography and radiochromatogram scanning revealed incorporation of radioactivity into the diacylglycerol (DAG) and hydrocarbon components as well as a third lipid fraction of unknown identity. Lipid transfer experiments were carried out using 14C-lipid HDLp-A and human low density lipoprotein (LDL) as donor/acceptor substrates and M. sexta lipid transfer particle (LTP) as catalyst. In control incubations lacking LTP, nearly all of the radiolabeled lipid remained associated with HDLp-A. LTP, however, induced a time-dependent vectorial transfer of radiolabeled lipid from HDLp-A to LDL. Lipid analysis of the LDL fraction, reisolated following the transfer reaction, revealed that labeled lipid components originally associated with HDLp-A were present in the acceptor LDL particles. The recovery of radiolabeled hydrocarbon associated with LDL demonstrates the capacity of LTP to facilitate transfer of these long chain, extremely hydrophobic, lipids and suggests LTP may function as a mediator of hydrocarbon transport and metabolism in vivo. When acceptor LDL particles were analyzed prior to complete transfer of HDLp-A-associated lipid it was observed that DAG was transferred preferentially during the initial stages of the reaction after which hydrocarbon transfer increased. This result suggests that LTP may have a lipid substrate preference for DAG versus hydrocarbon. Alternatively the observed preference for DAG may be a function of the relative accessibility of the substrates within the donor lipoprotein. In other experiments it was demonstrated that, unlike other lipids associated with HDLp-A, free fatty acid spontaneously transfers to LDL in the absence of lipid transfer catalyst.

Acetates↗
PubMed1990

Lipid transfer particle-induced transformation of human high density lipoprotein into apolipoprotein A-I-deficient low density particles.

Incubation of human high density lipoprotein (HDL) particles (density = 1.063-1.21 g/ml) with catalytic amounts of Manduca sexta lipid transfer particle (LTP) resulted in alteration of the density distribution of HDL protein such that the original HDL particles were transformed into new particles with an equilibrium density = 1.05 g/ml. Concomitantly, substantial amounts of protein were recovered in the bottom fraction of the density gradient. The LTP-induced alteration in HDL protein density distribution was dependent on the LTP concentration and incubation time. Electrophoretic analysis revealed that the lower density fraction contained apolipoprotein A-II (apoA-II) as the major apoprotein component while nearly all of the apoA-I was recovered in the bottom fraction. Lipid analysis of the HDL substrate and product fractions revealed that the apoA-I-rich fraction was nearly devoid of lipid (less than 1%, w/w). The lipid originally associated with HDL was recovered in the low density, apoA-II-rich, lipoprotein fraction, and the ratios of individual lipid classes were the same as in control HDL. Electron microscopy and gel permeation chromatography experiments revealed that the LTP-induced product lipoprotein population comprised particles of larger size (19.7 +/- 1.4-nm diameter) than control HDL (10.6 +/- 1.4-nm diameter). The results suggest that facilitated net lipid transfer between HDL particles altered the distribution of lipid such that apoprotein migration occurred and donor particles disintegrated. Similar results were obtained when human HDL3 or HDL2 density subclasses were employed as substrates for LTP. The lower surface area to core volume ratio of the larger, product lipoprotein particles compared with the substrate HDL requires that there be a decrease in the total exposed lipid/water interface which requires stabilization by apolipoprotein. Selective displacement of apoA-I by apoA-II or apoC, due to their greater surface binding affinity, dictates that apoA-I is preferentially lost from the lipoprotein surface and is therefore recovered as lipid-free apoprotein. Thus, it is conceivable that the structural arrangement of HDL particle lipid and apoprotein components isolated from human plasma may not represent the most thermodynamically stable arrangement of lipid and protein.

Animals↗
PubMed1990

Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta.

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0/100 mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KCl concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent S20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent S20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KCl which yielded a particle Mr = 887,000. Circular dichroism spectra of monomeric LTP sample revealed 6% alpha-helix, 49% beta-sheet, 7% beta-turn and 35% random coil while aggregated LTP contained 13% alpha-helix, 66% beta-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.

Animals↗
PubMed1990

Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein.

Insect plasma lipid transfer particle (LTP) catalyzes vectorial net transfer of diacylglycerol (DAG) from Manduca sexta larval high density lipophorin (HDLp-L) to human low density lipoprotein (LDL) producing an LDL of lower density and lipophorin subspecies of higher density. At equilibrium, a stable DAG-depleted very high density lipophorin species (density = 1.25 g/ml) is formed. Electrophoretic analysis of the substrate and product lipoproteins showed that apoprotein exchange or transfer between human LDL and lipophorin did not occur during the lipid transfer reaction. Facilitated net transfer of cholesteryl ester, free cholesterol, and phospholipid occurred to a much lower extent than DAG net transfer, indicating that under these conditions, LDL serves as a sink for lipophorin-associated DAG. This reaction, therefore, provides a method whereby the mass of lipid associated with human LDL can be modified in vitro without alteration of its apoprotein component. The DAG content of LDL increased in a linear manner with respect to LTP concentration and time during the initial phase of the reaction, demonstrating the utility of this system as a quantitative assay method for LTP-mediated net DAG transfer. When [3H]DAG-labeled LDL was prepared and employed in transfer experiments with unlabeled lipophorin, labeled DAG was recovered in the HDLp-L fraction. The amount of labeled DAG recovered in the HDLp-L fraction was dependent on the ratio of LDL to HDLp-L in the reaction. Thus, in this system, LTP-mediated DAG redistribution is bidirectional, suggesting that the final equilibrium distribution of lipid may be dictated by the properties of potential donor/acceptor lipoproteins rather than by an inherent particle substrate specificity of LTP.

Animals↗
PubMed1990

Lipid transfer between human plasma low-density lipoprotein and a triolein/phospholipid microemulsion catalyzed by insect hemolymph lipid transfer particle.

Lipid transfer between human plasma low-density lipoprotein (LDL) and an LDL-size microemulsion of triolein and phosphatidylcholine stabilized with human apolipoprotein A-I was catalyzed by the lipid transfer particle from hemolymph of the tobacco hornworm (Manduca sexta). Net transfer of phospholipid and triacylglycerol from the emulsion to LDL was observed and the apparent initial rates of transfer were dependent on the amount of catalyst. Net transfer of phospholipid mass was twice as much as that of triacylglycerol with respect to both the initial rate and the final equilibrium state. The final amount of net transfer of both lipids was dependent upon the initial ratio of LDL: microemulsion present in the incubation mixture up to 1:1 on the basis of phospholipid. The microemulsion lipid composition was maximally altered from an initial weight ratio of 1.09 +/- 0.08 (phospholipid/triolein) to 0.90 +/- 0.03 by this reaction. Further increase of LDL in the incubation caused neither further net transfer nor further change in the lipid composition of the microemulsion. The catalyst neither affected spontaneous transfer of free cholesterol between the emulsion and LDL nor enhanced cholesteryl ester transfer in this reaction system. As a result of the facilitated reaction, LDL gained a significant amount of phospholipid and triacylglycerol causing up to an 8% increase in core lipids and 14% in phospholipid. Some free cholesterol is recovered in the emulsions via spontaneous exchange. Transfer or exchange of apolipoproteins during the course of facilitated lipid transfer did not occur.

Animals↗
PubMed1990

Manduca sexta lipid transfer particle acts upon a lipoprotein to catalyze lipid and apoprotein disproportionation.

A novel reaction, catalyzed by Manduca sexta lipid transfer particle (LTP), transforms low density lipophorin (LDLp) into two distinct lipoprotein species. A population of LDLp particles serves as lipid donor or acceptor in LTP-catalyzed production of a very low density lipophorin (VLDLp) and a high density lipophorin (HDLp) product. The products result from facilitated net transfer of lipid mass from donor LDLp particles to acceptor LDLp particles. Transfer of apolipophorin III (apoLp-III) from donor to acceptor lipoprotein occurs during the reaction to produce a lipid- and apoLp-III-enriched VLDLp species and lipid- and apoLp-III-depleted HDLp species. The VLDLp produced in this in vitro reaction contains more lipid and apoLp-III than any previous lipophorin species reported and further demonstrates the scope of the lipid binding capacity of lipophorin. Lipid analysis and radiolabeling studies confirmed that unidirectional net transfer of lipid mass and apoLp-III from donor to acceptor occurs. When 3H-lipid-LDLp was used as substrate in the LTP-catalyzed disproportionation reaction the density distribution of radioactivity and protein provided evidence of vectorial transfer of diacylglycerol, phospholipid, and free fatty acids. Electron micrographs of the original LDLp population and of the LTP-induced product lipoprotein population provided further support for the interpretation derived from biochemical studies. This LTP-catalyzed disproportionation was observed only with apoLp-III-rich LDLp suggesting that the presence of increased amounts of this apoprotein dramatically affects the properties of the particle and appears to be directly related to the capacity of the lipoprotein to bind lipid.

Animals↗
PubMed1990

Studies of the morphology and structure of the plasma lipid transfer particle from the tobacco hornworm, Manduca sexta.

Morphological features of Manduca sexta plasma lipid transfer particle (LTP) have been investigated by electron microscopy. LTP was found to be an asymmetric particle with two major structural features: a roughly spherical head and an elongated, hinged tail. The hinge occurs approximately at the midpoint of the tail section with the two halves forming angles ranging from 30 degrees to 180 degrees. A molecular mass estimate of 1.4 x 10(6) daltons based on the dimensions of LTP suggests that multiple copies (two or three) of each of the three LTP apoproteins exist in the native complex. Limited digestion studies of LTP suggest that apoLTP-III is less susceptible to trypsin cleavage than apoLTP-I or -II, and therefore may be less exposed to the aqueous environment. Digestion for 1 h at a 1:50 trypsin-LTP protein ratio did not alter the flotation properties of LTP or its morphological features; thus, although significant proteolysis occurred, the particle retained its overall structure. Transfer activity, on the other hand, was affected by trypsin digestion with 30 +/- 14% inhibition of LTP activity occurring upon proteolysis at a 1:50 trypsin-LTP protein ratio. Treatment of LTP with phospholipase A2 resulted in the conversion of LTP-associated phosphatidylcholine and phosphatidylethanolamine to their corresponding lyso forms. Phospholipase A2 treatment did not, however, alter the SDS-PAGE profile, transfer activity, flotation pattern, or the microscopic features of LTP. These results suggest that the products of the phospholipase reaction remain associated with the particle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Facilitated diacylglycerol exchange between insect hemolymph lipophorins. Properties of Manduca sexta lipid transfer particle.

Manduca sexta hemolymph lipid transfer particle (LTP) is a very high density lipoprotein (d = 1.23 g/ml) containing 14% lipid and 5% carbohydrate. Each of three apoprotein components, apoLTP-I (Mr approximately 320,000), apoLTP-II (Mr = 85,000), and apoLTP-III (Mr = 55,000), is glycosylated. Carbohydrate analysis revealed the presence of mannose and N-acetylglucosamine in a ratio of 4.5:1. A native Mr greater than 670,000 was determined by pore limiting gradient gel electrophoresis. Lipid analysis of LTP revealed the presence of phospholipid, diacylglycerol (DAG), free fatty acid, and triacylglycerol. Rabbit polyclonal antibodies directed against LTP were obtained. Anti-LTP serum was employed in experiments which indicated the presence of LTP in larval and adult animals and confirmed that LTP was unrelated to other M. sexta hemolymph proteins and lipoproteins. A quantitative lipid transfer assay measuring facilitated DAG exchange between isolated M. sexta lipoproteins was established. The level of LTP-catalyzed exchange of DAG increased linearly with increasing time and protein during the initial phase of the reaction. Inclusion of anti-LTP serum in the assay inhibited facilitated DAG exchange. Experiments designed to determine if the LTP holoprotein is required for transfer or if a component of LTP is the active principle were performed. Incubation of [3H]DAG labeled high density lipophorin with substrate amounts of LTP resulted in incorporation of labeled DAG into LTP. Subsequent incubation of [3H]DAG-labeled LTP with unlabeled lipophorin resulted in exchange of DAG and the appearance of labeled DAG in lipophorin. Nitrocellulose-bound LTP apoproteins did not facilitate DAG exchange, and pretreatment of LTP with detergents resulted in loss of transfer activity. Extraction of LTP lipids with ethanol/ether also resulted in loss of activity. The results suggest that the lipid component of LTP may be important in the transfer reaction.

Animals↗
PubMed1988

Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very-high-density lipoproteins.

Facilitated diacylglycerol exchange between Manduca sexta [3H]diacylglycerol-labeled high-density lipophorin and Heliothis zea very-high-density chromolipoprotein was studied. M. sexta lipid transfer particle was employed in assays which measured exchange of [3H]diacylglycerol. Following incubations with lipid transfer particle, donor and acceptor lipoproteins were reisolated by density-gradient ultracentrifugation to determine facilitated exchange. The reaction was limited to diacylglycerol exchange, while donor or acceptor particle apoprotein exchange did not occur. Lipid analysis of donor and acceptor lipoproteins after the lipid-exchange reaction revealed that the labeled diacylglycerol remained unchanged. Lipid transfer particle-catalyzed diacylglycerol exchange was linear up to 0.3 micrograms lipid transfer particle protein in a standard assay and exchange occurred at a rate of 2.5 micrograms diacylglycerol min-1.micrograms-1 lipid transfer particle protein. The assay method was used to show that the hemolymph concentration of lipid transfer particle increased during development.

Animals↗
PubMed1988

An insect lipoprotein hybrid helps to define the role of apolipophorin III.

Insects transport lipid for flight in the form of diacylglycerol-rich low density lipoproteins (low density lipophorin (LDLp)). A hybrid LDLp has been produced in vitro by using Locusta migratoria fat body, locust high density lipophorin, locust adipokinetic hormone, and Manduca sexta apolipophorin III (apoLp-III). The hybrid is similar in size and density to locust LDLp, contains several molecules of M. sexta apoLp-III, and lacks locust apoLp-III, as shown by immunochemical methods. Under the same conditions an apoLp-III from Thasus acutangulus is poorly incorporated into the locust lipoprotein. The role of apoLp-III as a recognition signal/activator of flight muscle lipoprotein lipase was assayed with labeled hybrid LDLp produced in vitro using M. sexta apoLp-III radiolabeled with 35S. In addition, hydrolysis of diacylglycerol was determined with lipid-labeled hybrid LDLp produced in vitro using [U-14C]glycerol incorporated into the diacylglycerol moiety. In vitro incubations of the labeled hybrid LDLp with L. migratoria flight muscles show that the lipase efficiently utilizes hybrid LDLp as a substrate and demonstrate that the carbohydrate moiety of locust apoLp-III (which is lacking in the M. sexta protein) is not required for interaction with the lipase. It also suggests that specific antigenic determinants of L. migratoria apoLp-III are not required for lipase activation since M. sexta apoLp-III lacks immunological cross-reactivity with L. migratoria apoLp-III.

Animals↗
PubMed1987

Purification and properties of a very high density lipoprotein from the hemolymph of the honeybee Apis mellifera.

A larval-specific very high density lipoprotein (VHDL) has been isolated from the hemolymph of the honeybee Apis mellifera. VHDL was isolated by a combination of density gradient ultracentrifugation and gel filtration. The purified protein is a dimer of Mr 160,000 apoproteins as shown by chemical cross-linking with dimethyl suberimidate. N-Terminal sequence analysis indicates that the two polypeptide chains are identical. The holoprotein contains 10% lipid by weight and 2.6% covalently bound carbohydrate. A native Mr 330,000 species was obtained by gel permeation chromatography. Antiserum directed against VHDL was used to show that VHDL is distinct from other hemolymph proteins and appears to constitute a novel lipoprotein of unknown function. However, the lipoprotein is present in high amounts in hemolymph only at the end of larval life, suggesting a potential role in lipid transport and/or storage protein metabolism during metamorphosis.

Amino Acid Sequence↗
PubMed1987

The role of apolipophorin III in in vivo lipoprotein interconversions in adult Manduca sexta.

Sustained flight in the moth, Manduca sexta, necessitates lipid mobilization and transport to flight muscle, a process mediated by the adipokinetic hormone. An adult specific high density lipophorin (lipoprotein, HDLp-A, Mr = 7.68 X 10(5)) accepts diacylglycerol from the fat body, increasing in size and decreasing in density, to give a low density lipophorin (lipoprotein, LDLp, Mr = 1.56 X 10(6)). During this process, several molecules of the small apolipoprotein, apolipophorin III (apoLp-III), are added to the two molecules originally present in HDLp-A. A study of the time course of adipokinetic hormone-induced loading of diacylglycerol onto HDLp-A, using the analytical ultracentrifuge and gel filtration, suggests that a lipoprotein of density intermediate between HDLp-A and LDLp was formed transiently. Analysis of lipoproteins separated by density gradient ultracentrifugation in the course of the loading process indicates that apoLp-III is added more rapidly than diacylglycerol and that it changes its conformation on the surface as more diacylglycerol is added. Taken together with the known properties of apoLp-III, a prolate ellipsoid with an axial ratio of 3, we suggest that initially apoLp-III adds to the expanded hydrophobic surface of the lipoprotein with its short axis parallel to the surface and that apoLp-III subsequently unfolds to cover a greater area of hydrophobic surface. Exchange experiments with labeled apoLp-III showed that the two apoLp-III molecules in HDLp-A do not exchange with free apoLp-III, even when the lipoprotein passed through a loading and unloading cycle, suggesting a structural role for apoLp-III in HDLp-A.

Animals↗
PubMed1987

Purification of very high density lipoproteins by differential density gradient ultracentrifugation.

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.

Animals↗
PubMed1986

Lipid transfer protein from Manduca sexta hemolymph.

A hemolymph lipid transfer protein (LTP) was isolated from the tobacco hornworm, Manduca sexta. LTP catalyzes net lipid transfer between isolated hemolymph lipoproteins in vitro. An isolation procedure employing density gradient ultracentrifugation and gel permeation chromatography produced a purified protein. LTP is a very high density lipoprotein with a particle Mr greater than 500,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that LTP is comprised of two apoproteins: apoLTP-I (Mr approximately 320,000) and apoLTP-II (Mr approximately 85,000). LTP may have a physiological role in altering the lipid content and composition of the major hemolymph lipoprotein, lipophorin.

Animals↗
PubMed1986

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta.

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.

Animals↗