Insect Biochemistry最新文献

The male accessory glands of Locusta contain factors which stimulate oviduct contraction. From an extract of 4400 gland masses, a myotropic peptide (Lom-AG-myotropin II) was isolated by HPLC. Sequencing revealed the following sequence: Ala-His-Arg-Phe-Ala-Ala-Glu-Asp-Phe-Gly-Ala-Leu-Asp-Thr-Ala. Chemical synthesis confirmed that this peptide is active in the acid form instead of the amide form as other presently known myotropic peptides. The sequence does not resemble that of any peptide isolated from Locusta or other insects. The myotropin is active on the oviduct of Locusta migratoria but not on the oviduct of Leucophaea maderae. The myotropic activity can be detected on the hindgut of both insects but at much higher peptide concentrations.

Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the M_r value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent K_m value of 0.28 × 10⁻³ M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca²⁺, Na⁺ and Fe³⁺ and was stimulated by Zn²⁺, Mn²⁺ and Mg²⁺.

The arylphorins and LSP-2 like polypeptide were detected by immunoblotting analysis during development in the integument of C. capitata. In vitro translation of total RNA from fat body and integument during pupariation, clearly revealed that the polypeptides under consideration were exclusively synthesized in the fat body. Furthermore, in vitro experiments demonstrated that radiolabeled arylphorins and LSP-2 like polypeptide were taken up by the integument, in an undegraded state. Immunofluorescence experiments in cross sections of wandering stage larvae and white pupae revealed that the LSP-2 like polypeptide was mainly localized in the epidermal cells, and a very weak signal was also given by the cuticle. Furthermore, the presented results indicated that a small portion of the extracted proteins exist in high molecular weight aggregate(s).

A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M_r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K_m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.

A prophenoloxidase was purified from blood cells of the crayfish Pacifastacus leniusculus. The purified proenzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had a molecular mass of 76 kDa under both non-reducing and reducing conditions. The crayfish prophenoloxidase was a glycoprotein, with an isoelectric point of about 5.4.

A 36 kDa serine proteinase, isolated and purified from crayfish blood cells (Aspán et al., 1990b, Insect Biochem.20, 709–718), could convert the 76 kDa prophenoloxidase to phenoloxidase by an apparent proteolytic cleavage, since the molecular masses of two active enzymes, phenoloxidases, were 60 and 62 kDa. A commercial serine proteinase, trypsin, activated prophenoloxidase to phenoloxidase, and as a result a 60 kDa protein was produced.

In the blood cells of crayfish four serine proteinases or ³H-DFP binding proteins are present, with masses of 36, 38, 50 and 67 kDa. However, ³H-DFP labelling of proteins in blood cells lysate, prepared in its inactive form, only yielded labelled bands of 50 and 67 kDa, whereas addition of an elicitor to prophenoloxidase system activation, a β-1,3-glucan, resulted in the appearance of four ³H-DFP labelled proteins, with molecular masses of 67, 50, 38 and 36 kDa, respectively. Thus, the 36 kDa endogenous serine proteinase, the prophenoloxidase activating enzyme, ppA, may be present as an inactive precursor in crayfish blood cells. The 38 and 36 kDa proteinases could both cleave the chromogenic peptide S-2337 [Bz-Ile-Glu-(γ-O-Piperidyl)-Gly-Arg-p-nitroaniline], and specifically bind prophenoloxidase.

These results show that crayfish prophenoloxidase, the terminal enzyme of the prophenoloxidase activating cascade, a proposed defence pathway in arthropod blood, can be converted to active enzyme by an apparent proteolytic cleavage, not only by a commercial proteinase, but also by an endogenous serine type proteinase.

The ability of a baculovirus vector system to produce insect eclosion hormone (EH) was investigated by insertion of a Manduca sexta cDNA encoding EH into the genome of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) such that transcription was under the control of a strong, modified polyhedrin promoter. Two polypeptides, approx. 6.0–6.5 kDa, were synthesized in and secreted from recombinant virus-infected cells but not wild-type (wt) virus-infected cells. Both polypeptides were immunoprecipitated by polyclonal antiserum directed againnst natural M. sexta EH. Immunoblotting revealed only a single polypeptide, suggesting that only one form is stable in the expression system. The size of this polypeptide and its elution from a reverse phase HPLC column indicate that this polypeptide is similar, if not identical, in structure to natural EH. Bioassay data revealed that biologically active EH was synthesized and secreted at high levels (e.g. 10 μg of active polypeptide per 10⁶ cells). Thus, the baculovirus expression system is an excellent source of EH for further studies of this unique insect neurohormone.

Immunological properties and content changes of cyanoprotein (CP) were investigated in the bean bug, Riptortus clavatus. Anti-CPegg serum was prepared by immunizing a rabbit with CP purified from eggs (CPegg). In the Ouchterlony double diffusion test, the precipitin line between CPegg and anti-CPegg serum fused with that of non-diapause and diapause female hemolymph and anti-CPegg serum. Rocket immunoelectrophoresis (RIE) using anti-CP serum showed two types of rockets (A and B) depending on the samples. Namely, CPegg and non-diapause female adult hemolymph formed A rockets (heavy-stained with Coomassie Brilliant Blue) and early diapause female adult hemolymph formed B rockets (light-stained), but hemolymph from fifth instar nymphs formed both A and B rockets. Both rockets A and B were demonstrated by SDS-PAGE analysis of the precipitin lines to be formed from the same CP subunit (MW, 76,000). CP-1, 2 and 3 bands from native PAGE of nymphal hemolymph formed A rockets and CP-4 formed B rockets. The contents of CP-A (CP-1 to 3) and CP-B (CP-4) were separately determined by measuring the sizes of rocket A and B. CP-A and CP-B content were demonstrated to increase during the development of the last instar nymph and decrease at adult emergence by RIE analysis of non-diapause female whole body extracts. CP-B is predominant in the nymphal stage. In the early adult stage (day 2 and 3 after emergence), neither CP-A nor CP-B were detected. Only CP-A appeared again at day 4 after emergence and increased during development and vitellogenesis of non-diapause females.

Sclerotized oothecae from four species of cockroaches, Periplaneta americana, P. fuliginosa, Blatta orientalis and Blattella germanica, were examined by solid-state ¹³C-nuclear magnetic resonance and chemical analyses. The oothecae were composed of protein, water, calcium oxalate, diphenolic compounds, lipid, and uric acid. Calcium oxalate was the major soluble component in egg cases of P. americana, P. fuliginosa, and B. orientalis. Oothecae of B. germanica had approx. 10-fold less calcium oxalate and extractable diphenols than the other species. The major diphenolic compound extracted in cold dilute perchloric acid was 3,4-dihydroxybenzoic acid. Exuviae from P. americana, B. germanica, Gromphadorhina portentosa, Blaberus craniifer, and Leucophaea maderae also were examined by solid-state ¹³C-NMR. They contained protein, diphenols, and lipid, as well as chitin, which accounted for 30–42% of the organic content, depending upon the species.

Mechanical disruption of day 7 fifth instar Manduca sexta larval prothoracic glands prior to in vitro incubation with [³H]cholesterol resulted in a dramatic 10-fold increase in its conversion to [³H]7-dehydrocholesterol (20–50%), when compared to intact gland incubations (2–5%). Both procedures resulted in a 0.5–2% conversion to [³H]ecdysteroids. Endogenous cholesterol levels also decreased by this same 20–50% during the incubation, suggesting that the added tracer [³H]cholesterol was equilibrated with the total endogenous cholesterol pool. Glands from earlier fifth instar larvae were capable of similar conversion of [³H]cholesterol to [³H]7-dehydrocholesterol but without concomitant conversion to [³H]ecdysteroids, while in day 7 glands, conversion to [³H]ecdysteroids was temporally correlated with both the in vitro secretory activity of intact glands and the endogenous hemolymph ecdysteroid titer. These data suggest that the rate-limiting step in ecdysteroid biosynthesis occurs after the synthesis of 7-dehydrocholesterol. In addition to 7-dehydrocholesterol and ecdysteroids, four intermediate polarity metabolites were detected following the incubation of disrupted prothoracic glands. One, M1, appears to be an immediate precursor of ecdysone and 3-dehydroecdysone. Another, M3, while not a precursor of the ecdysteroids, may be a degradation product of a proposed epoxide intermediate of 7-dehydrocholesterol. A hypothetical scheme for the biosynthesis of ecdysteroids from cholesterol is presented.