Biochemical and immunological properties of different electrophoretic forms of juvenile hormone esterase from Trichoplusia ni (Hübner)

The two major electrophoretic forms (pI 5.5, 5.3) of juvenile hormone esterase were independently isolated from hemolymph of larval Trichoplusia ni. A simple and rapid preparation procedure of poly(ethylene glycol) precipitation, Sephadex gel filtration and chromatofocusing is described. Analytical isoelectric focusing showed only one peak of juvenile hormone esterase activity in the respective purified samples, whereas there were four (two major) such peaks in the hemolymph. The amino acid composition of the two forms was similar. The comparison of peptides obtained after protein fragmentation by cyanogen bromide showed that juvenile hormone esterases A and B were very similar, although definitely not identical, in amino acid sequence. The immunological comparisons of juvenile hormone esterases suggested that the number of polyclonal antibody binding sites on both forms was the same. There were no detected differences between immunoreactive properties of juvenile hormone esterase from the hemolymph of different stages of larval maturation. The influence of the active site of the enzyme on its antigenic properties was studied by immunocompetition. The inactive, heat-denatured juvenile hormone esterase can only partially protect against inhibition of its activity by the antibodies, whereas an organophosphate inhibitor which covalently binds to the catalytic center of the enzyme did not change the immunoreactive properties in comparison to active juvenile hormone esterase from hemolymph. These data show that heat-denatured juvenile hormone esterase has lost at least one or more epitopes, but the catalytic site of the enzyme is distinct from the epitopes.     

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm,Spodoptera litura

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.     

Identification of specific interaction of juvenile hormone binding protein with isocitrate dehydrogenase

Juvenile hormone (JH) is essential for multiple physiological processes: it controls larval development, metamorphosis and adult reproduction. In insect hemolymph more than 99 % of JH is bound to juvenile hormone binding protein (JHBP), which protects JH from degradation by nonspecific hydrolases and serves as a carrier to supply the hormone to the target tissues. In Galleria mellonella hemolymph, JHBP is found in a complex with lipid-binding high molecular weight proteins (HMWP) and this interaction is enhanced in the presence of JH. In this report, we present studies on the interaction of JHBP with low molecular weight proteins (LMWP) in the hemolymph. Using ligand blotting we found that JHBP interacts with a protein of about 44 kDa. To identify the protein that preferentially binds JHBP, a LMWP fraction was applied to a Sepharose-bound JHBP and, after washing, the column was eluted with free JHBP acting as a specific competitor or with carbonic anhydrase as a negative control. The eluted proteins were separated by SDS/PAGE and analyzed by mass spectrometry. Isocitrate dehydrogenase was identified as a component of the supramolecular complex of JHBP with hemolymph proteins.     

Synthesis and binding affinity of an iodinated juvenile hormone

The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural 3H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated [125I]12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added 125I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of [125I]12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.     

Insect apolipophorin III. Purification and properties

The hemolymph of adult Manduca sexta (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin III. Apolipophorin III was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin III was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin III is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr = 34,000.     

Purification,properties, and titer of a hemolymph trophic factor in larvae and pupae of Manduca sexta

Purification of a hemolymph protein (hemolymph trophic factor, or HTF) from last instar larvae of Manduca sexta was achieved using Sephadex G15-120 gel filtration and DEAE anion exchange chromatography. Homogeneity was visualized using SDS gel electrophoresis and ampholytic chromatofocusing. HTF was estimated to be a tetrameric protein with a molecular weight of 286 K and a Stokes' radius of 55.3 × 10⁻⁸ cm by agarose bead gel filtration; chromatofocusing suggests an isoionic point > 10. Polyclonal antibodies to HTF were prepared in rabbits and an ELISA was developed. The ELISA was used to titer HTF during the last larval instar and day 1 and 14 of the pupal stage and estimates a maximum of 1.5 mg/ml larval hemolymph on day 6 with a smaller larval peak of 0.75 mg/ml at day 3 and titers of 0.70 and 0.35 mg/ml on the 2 pupal days, respectively. ELISA of aqueous extracts of larval fat body, epidermis, and cuticle demonstrate that HTF comprises nearly a third of the soluble fat body protein and is a lesser component of epidermis and cuticle. The physiological role of HTF has not yet been determined.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

Regulation of glutamine metabolism during the development of Bombyx mori larvae

Waste ammonia is re-assimilated into amino acids via the amide group of glutamine and the amino group of glutamate (i.e. through glutamine synthetase/glutamate synthase pathway) for silk synthesis in the silkworm, Bombyx mori, in the last larval stadium. Glutamine concentration in hemolymph gradually decreased with the progress of the fifth instar and it remained at very low levels during the spinning stage, then followed by a sharp increase at the larval-pupal ecdysis. The changes in glutamine synthetase (GS) activity in silkworm tissues were relatively small through the larval development, while the changes in glutamate synthase (GOGAT) activity, especially in the posterior silk glands, were more drastic. In addition, activities of GOGAT in the tissues were much higher than those of the other enzymes involved in glutamine utilization, suggesting that glutamine pool was regulated mainly by the changes in GOGAT activity. Western blot analysis indicated that the changes in GOGAT protein level correlated with the changes in GOGAT activity. Topical application of a juvenile hormone analogue, methoprene, induced an accumulation of glutamine in the hemolymph of the fifth instar larvae. The levels of GOGAT protein and activity in the tissues of the methoprene treated larvae were much lower than those of the control larvae, whereas the methoprene treatment had no effect on the levels of GS activity. In conclusion, GOGAT expression promoted by reduction of juvenile hormone titer is quite important for enhanced utilization of nitrogen for synthesis of silk protein during the last larval instar.     

Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization

A juvenile-hormone-binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogeneous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32,000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm epsilon = 2.34 X 10(4) M-1 cm-1, and is composed of a single polypeptide chain. Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues. The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 X 10(-8) M; JH II, 7.2 X 10(-8) M; JH III, 47 X 10(-8) M. The JHBP binds (10R, 11S)-JH II enantiomer with 2.3-times higher affinity then (10S, 11R)-JH II enantiomer. The pH optimum of binding is 7.0.     

A carrier lipoprotein for juvenile hormone in the haemolymph of Locusta migratoria

In the haemolymph of adult female locusts six different lipoprotein fractions have been demonstrated by means of isoelectric focusing. One of these binds injected ³H-Cecropia juvenile hormone. The carrier protein is a yellow lipoprotein with a molecular weight of approximately 220,000 daltons and an isoelectric point of pH 6·8. The binding of the hormone to the protein is stable during gel filtration over Sephadex G-25 and during dialysis for 24 hr against phosphate buffer pH 7·0.The hormone is quickly metabolized in the locusts. In the haemolymph were found more polar compounds such as 10-epoxy-7-ethyl-3,11 dimethyl-2,6-tridecadienoic acid and the corresponding dioldienoic acid.Both compounds were not bound by the pH 6·8 carrier lipoprotein under in vivo conditions.     

Purification and characterization of an iron-binding protein from the blue crab (Callinectes sapidus)

The presence of an iron-binding protein in the hemolymph of the blue crab (Callinectes sapidus) was detected by gel filtration of 59Fe-labeled hemolymph. The iron-binding protein was purified to homogeneity by ion exchange chromatography. 2. This protein has a mol. wt of 155,000 and consists of a single polypeptide chain with an isoelectric point of 5.0. 3. Analysis of the iron-loaded protein indicates that it has a high affinity for iron and the capacity to bind approximately 10 atoms iron/molecule protein. 4. The isolation of a specific iron-binding protein from the blue crab (Callinectes sapidus) provides additional support for the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins (hemoglobin and hemerythrin) as a means for oxygen transport.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Viresin. A novel antibacterial protein from immune hemolymph of Heliothis virescens pupae.

Immune hemolymph was collected from fifth instar larvae and 1-day-old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N-terminal amino acid sequence of 12-kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12-kDa protein was negative for immunoblotting with anti-synthetic cecropin B antibody. We have named the novel 12-kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram-negative bacteria including E. cloacae but not against Gram-positive bacteria.     

Purification and characterization of an anti-apoptotic protein isolated from Lonomia obliqua hemolymph

Previously it was reported that supplementation of insect cell culture with Lonomia obliqua hemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58-63). In this work the anti-apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf-9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti-apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf-9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliqua hemolymph is due to the presence of potent anti-apoptotic factors.     

Juvenile hormone esterase is a biochemical anti-juvenile hormone agent

Juvenile hormone esterase, purified by affinity chromatography from the larval hemolymph of Manduca sexta in the fifth stadium, was injected into larvae of the same species in the earlier stadia resulting in a blackening of the cuticle following ecdysis to the next larval stadium. This anti-juvenile hormone response was dose-dependent for an injection in the second, third or fourth stadium. Cuticular blackening was prevented by treating larvae with the juvenoid epofenonane. Larval response to injected juvenile hormone esterase also varied with the time of injection within a single stadium, having a maximum effect for injections at the time of head capsule slippage. Juvenile hormone esterase activity measured from the hemolymph after injection of larvae in the second stadium decreased over an 11 h time-course. Because the anti-juvenile hormone effects resulting from a single injection of juvenile hormone esterase were dependent on the time of injection, it appears that when juvenile hormone biosynthesis is active in the insect, the duration of enzyme activity limits the anti-juvenile effects that can be induced.     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

Purification of Major Hemolymph Protein of Great Wax Moth, Galleria mellonella

Major hemolymph protein (MHP) was purified from larval hemolymph of Galleria mellonella by KBr density gradient ultracentrifugation, ion exchange chromatography (DEAE‐Trisacryl M), YM‐50 ultrafiltration and gel permeation chromatography (Sephadex G‐100). MHP is composed of two subunit (MHP‐1 and MHP‐2). The molecular weights of each subunit were determined (MHP‐1 = 86 kDa and MHP‐2 = 84 kDa). MHP is present in both hemolymph and fat body during developmental stages, indicating this protein is carrying out some functions different from other major protein such as storage protein and lipophorin.     

Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.     

Binding of juvenile hormone III to lipophorin from the american cockroach Periplaneta americana

Whole hemolymph from the American cockroach, Periplaneta americana, efficiently binds juvenile hormone (JH) III and to a lesser extent JH-I and 10, 11-epoxyfarnesyl diazoacetate (EFDA). The dissociation constants for racemic JH-III and EFDA are 30 ± 2 nM and 1.0 μM, respectively. Isolated lipophorin also binds [³H]JH-III and to a lesser extent JH-I. Other proteins from the hemolymph do not bind JH-III. Binding of JH-III to lipophorin is enantioselective. The dissociation constant, measured with a 92% 10R and 8% 10S mixture, is 21 ± 2 nM. Each lipophorin molecule contains one specific binding site for JH-III. It is concluded that lipophorin is the JH-III-specific transport protein in the hemolymph of the American cockroach. By a combination of photoaffinity labelling and gradient electrophoresis with sodium dodecyl sulphate on polyacrylamide gel, we showed that the JH-III-specific binding site is probably located on apolipophorin I.     

Development and partial characterization of monoclonal antibodies to the hemolymph juvenile hormone binding protein of Manduca sexta

Murine monoclonal antibodies were made against the hemolymph juvenile hormone binding protein (JHBP) of Manduca sexta. Binding studies in conjunction with Western blot analysis of native and sodium dodecyl sulfate gels confirmed that antibodies from 10 hybridoma lines interacted with the juvenile hormone binding protein. The pattern of cross-reactivity among the hybridoma lines suggests that different epitopes are recognized. The cross-reactivity pattern for monoclonal antibody 9 suggested a common epitope in three different hemolymph proteins: JHBP, insecticyanin and a 40–45 kDa protein. Western blot analysis of a two-dimensional gel using monoclonal antibody 6 revealed interaction with JHBP and with several proteins that may be precursors or degradation products of the binding protein. An enzyme-immunoassay was developed that detects JHBP in the hemolymph at nanogram levels.