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.     

Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Manduca sexta

Lipoprotein biosynthesis in larvae of the tobacco hornworm (Manduca sexta) was investigated. By immunoblotting, it was shown that the apoproteins are present in the fat body, but not in the midgut. Fat body incubated in vitro with [35S]methionine secreted labeled apoproteins. However, when the density of the secreted particle was determined, it was found at 1.24-1.28 g/ml instead of 1.15 g/ml, which is the density of the circulating lipoprotein. Lipid analysis of immunoprecipitated lipoprotein secreted by the fat body showed a phospholipid/diacylglycerol ratio of 8.3 rather than 0.9, the ratio found in the circulating lipoprotein. When labeled oleic acid or triolein was fed to larvae, it was found that greater than 98% of the label in the circulating lipoprotein was in diacylglycerol. In studies using animals raised on a fat-free diet, it was shown that the circulating lipoprotein has properties comparable to those of the material secreted in vitro by the fat body and that this diacylglycerol-poor particle can be converted to the normal lipoprotein by feeding a bolus of triolein. These data support the hypothesis that the fat body makes and secretes a "nascent" lipoprotein which contains apoproteins and phospholipid, but is devoid of diacylglycerol. The diacylglycerol is then picked up from the midgut to complete assembly of the mature circulating lipoprotein.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Crystallization of insecticyanin from the hemolymph of the tobacco hornworm Manduca sexta L. in a form suitable for a high resolution structure determination

Insecticyanin, a blue biliprotein from the tobacco hornworm Manduca sexta, has been crystallized in a form suitable for a high resolution x-ray analysis. The crystals grow by vapor diffusion against solutions of polyethylene glycol 8000 at pH 5.5. They belong to the space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 115.0 A; c = 71.1 A. Insecticyanin is believed to be a tetramer in solution; there are two subunits per asymmetric unit. The crystals diffract to at least 2.2 A resolution and appear reasonably resistant to radiation damage.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

Resistance to phagocytosis by group A streptococci: failure of deposited complement opsonins to interact with cellular receptors

The previous finding that phagocytosis-resistant M+ group A streptococci bear quantities of C3 which are sufficient for phagocytosis of their M- derivatives was investigated at two levels. It was first established that the C3 associated with M+ streptococci was not able to promote adherence to cells bearing the complement receptors CR1 and CR3 under conditions in which M- streptococci readily attached. The molecular form of C3 bound to M+ and M- streptococci was then defined by adding 125I-C3 to serum used for opsonization. C3 eluted from the bacteria by chaotropic and hydrolytic agents was analyzed by SDS-PAGE, and revealed that both cell types bound the opsonic forms of C3, C3b, and iC3b. Furthermore, approximately 80% of the C3b and iC3b associated with both cell types was covalently bound to a surface component, although most of the C3 bound to M+ streptococci was detergent-extractable, whereas greater than 50% of that bound to M- streptococci was not. These findings demonstrate that the M+ surface is interfering with the receptor binding of deposited C3b and iC3b, and that this contributes to resistance to phagocytosis by these organisms.     

Determination of Endogenous Indole-3-Acetic Acid in Plagiochila arctica (Hepaticae)

Endogenous indole-3-acetic acid (IAA) was found in axenically cultured gametophytes of the leafy liverwort, Plagiochila arctica Bryhn and Kaal., by high-performance liquid chromatography with electrochemical detection. Identification of the methylated auxin was confirmed by gas chromatography-mass spectrometry. Addition of 57 micromolar IAA to cultures increased relative production of ethylene. This is the first definitive (gas chromatography-mass spectrometry) demonstration of the natural occurrence of IAA in a bryophyte.     

Cleavage of Rabbit Myelin Basic Protein by Plasmin: Isolation and Identification of the Major Products

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.