Insect Biochemistry最新文献

Previous studies reviewed here have indicated that juvenile hormone (JH) specifically binds to a 29 kDa protein in epidermal nuclei from Manduca sexta larvae. Also, a 29 kDa nuclear protein that showed the same developmental pattern bound specifically to a larval cuticle gene LCP14. These results indicate that the 29 kDa nuclear protein is likely a JH receptor. Two retinoids (SRI 5942-64 and Ro 13-6298) were found to be weak JH mimics with ED₅₀s 60–100 times higher than that of JH III in the black Manduca larval bioassay. A genomic clone (Manduca “RAR”) then was isolated using the human retinoic acid receptor (hRAR) cDNA and the homologous region was sequenced. Thirteen out of 14 amino acids constituting the C-terminal half of the second zinc finger were identical in Manduca “RAR” and hRAR. Manduca “RAR” selected two mRNAs (3.8 and 4.5 kb) that are expressed at the peaks of the ecdysteroid titer during both the larval and the pupal molts, but not during the intermolt periods. When pieces of integument from day two 4th instar larvae were cultured with 4 × 10⁻⁶ M 20-hydroxyecdysone (20HE), Manduca “RAR” mRNA increased 13–15-fold by 6 h, then decreased after 12 h in the continuous presence of 20HE. The presence of 3 × 10⁻⁶ M JH slowed the rate of induction by 20HE. Thus, the “RAR” gene product is likely not the 29 kDa JH receptor but rather a transcriptional regulatory factor whose presence during a molt is modulated by JH.

Protein phosphatase activity in tick salivary glands was inhibited by heat-stable protein(s) from tick salivary glands as well as by an inhibitor protein from rabbit skeletal muscle. Inhibitor activity was increased after phosphorylation of inhibitor proteins with the catalytic subunit (C) of cyclic AMP-dependent protein kinase and ATP. C inhibited protein phosphatase activity of the partially purified enzyme, while purified cyclic AMP-dependent protein kinase inhibitor protein prevented inhibition of tick salivary gland protein phosphatase by C suggesting that the inhibitor phosphoprotein coelutes with the partially purified enzyme. A soluble heat-stable protein with a molecular weight of approx. 26 kDa was phosphorylated by C, suggesting that a protein phosphatase inhibitor protein similar to inhibitor-1 in mammalian tissue, is present in tick salivary glands.

Vitellogenin (Vg), the yolk precursor protein and its product vitellin (Vn) have been identified in hemolymph and fat body of females and mature oocytes in the millipede Spirostreptus asthenes employing double immunodiffusion technique. These two proteins are absent in males indicating that they are female specific. Immunoelectrophoresis has shown that there is only one vitellogenic protein present in S. asthenes. Vg and Vn were isolated by gel filtration. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that Vg and Vn are glycolipoproteins. Vg contains 48.8% protein, 2.2% carbohydrate and 48.9% lipid. Vn is comprised of 52% protein, 2.3% carbohydrate and 45.4% lipid. The lipid components of Vg and Vn include mainly phospholipids such as phosphatidic acid, sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine and cholesterol. On SDS-PAGE analysis both Vg and Vn yielded five sub units each. The molecular weight of the sub units of Vg was found to be 135, 115, 105, 73 and 56 kDa and those of Vn were 125, 110, 100, 68, and 53 kDa. The vitellogenic system of S. asthenes resembles that of insects. The phylogenetic relationship of the vitellogenic system of this millipede with other arthropod groups is discussed.

A sensitive assay for kynurenine transaminase activity (E.C. 2.6.1.7) based on rapid separation of the reaction product by high performance liquid chromatography (HPLC) has been developed. Drosophila sordidula extracts have been assayed by this new method and this is the first time that kynurenine transaminase activity has been demonstrated in Drosophila. The method of assay developed can be extended to any other organism. Kynurenine and 3-hydroxykynurenine were both used as substrates, and they were transaminated to kynurenic acid and xanthruenic acid, respectively. HPLC is used to separate and quantitate these reaction products from all other components in the reaction mixture.

In crude extracts from Drosophila, the reaction requires pyridoxal 5′-phosphate and an amino acid acceptor. The enzyme activity showed a maximum at 47°C and pH 8.0 with kynurenine and pyruvic acid as substrates. Transaminase activity was present in both head and body, nevertheless the specific activity was higher in the former. In bodies, pyruvic acid was the best amino acceptor whereas in heads it was α-oxoglutaric acid. The variation of kynurenine transaminase during development of D. sordidula showed, in the larval and pupal stages, activity levels practically constant and much lower than those found in the adult. This seems to suggest a preferential role of this enzyme in the metabolism of intermediates in the biosynthesis of ommochromes.

Recent developments in automated peptide microsequencing, liquid secondary-ion and electrospray mass spectrometry enable unambiguous primary structure determinations of minute amounts of biological material. We have used these methods in combination to characterize the predominant peptides from HPLC eluates of aqueous extracts of corpora cardiaca from adults of Locusta migratoria. Among the molecules or families of molecules clearly predominating in the extracts, we had previously characterized novel peptides (Hietter et al., 1989, 1990), and we recently identified three structurally-related, cysteine-rich, 8–9 kDa peptides. We present in this paper their complete structure determination. The amino acid sequence of these peptides is superimposable to that of neuroparsins isolated as dimers by Girardie et al., 1989. However, our experimental data lead us to propose that these molecules are monomers containing six intramolecular disulfide bridges.

Protein, carbohydrate, free amino acid, lipid, RNA levels, and electrophoretic changes in the protein profile were determined in the eggs of the water scorpion, Laccotrephes griseus, during normal embryonic development. The protein levels remain lower and relatively constant in the eggs of 0, 1 and 2 days of age, while in the eggs of older groups, i.e. between 2 and 6 days, a marked increase in the protein level occurs. Then its level declines. The RNA content shows a rise up to the day 6 stage, later it declines sharply, indicating an increase in the degree of synthetic activity that takes place during such period of embryonic development. Electrophoretic and densitometric analysis show the qualitative and quantitative changes of yolk protein reflecting the utilization of already existing proteins as well as the appearance of new proteins.

Water content increases gradually as development proceeds. There is a steady depletion of carbohydrate and lipid during the course of embryonic development. The nature of yolk components as well as their preferential utilization during embryogenesis has been discussed in relation to the generally accepted view that protein serves as the source for the embryonic metabolism in aquatic insects does not hold good for L. griseus and other freshwater insects.

Novel aspects of the metabolism of polyunsaturated fatty acids (PUFAs), precursors of prostaglandins and other eicosanoids, in insects are reviewed. A number of insect species are able to produce linoleic acid, 18:2 ($\text{n-6}$), de novo, a fatty acid that was previously believed to be essential for all animals. These insect species are unique among animals in that they possess a Δ12 desaturase which converts oleic acid, 18:1 ($\text{n-9}$) to 18:2 ($\text{n-6}$) and makes them nutritionally independent of plant derived polyunsaturated fatty acids (PUFA). The potential role of microorganisms in linoleate production was ruled out by studies using isolated tissue under axenic conditions from the house cricket, Acheta domesticus, an insect which does not contain intracellular microorganisms. The results showed that it is insect tissue that contains the Δ12 desaturase. This enzyme has been charactized from the house cricket and the American cockroach. In contrast to the plant Δ12 desaturase, which converts 18:1 esterified in a phospholipid as substrate to 18:2 ($\text{n-6}$), the insect Δ12 desaturase uses oleoyl-CoA as substrate. A number of insect species, including representatives of both those that do and do not produce linoleate, elongate and desaturate 18:2 ($\text{n-6}$) and 18:3 ($\text{n-3}$) to 20:4 ($\text{n-6}$) and 20:5 ($\text{n-3}$), respectively. A conspicuous exception to this are mosquitoes, which require 20:4 ($\text{n-6}$) or structurally related fatty acids in their diet. A number of insect species have been shown to metabolize 20:4 to prostaglandins and other eicosanoids.

The wing epidermis of Manduca sexta has 8 prominent proteins of molecular weights ranging from 18,000 to 55,000, the in vitro phosphorylation of which is enhanced significantly by cAMP. The level of protein phosphorylation during pupal-adult development can be correlated with the changing hemolymph ecdysteroid titer. These protein substrates are not limited to the wing epidermis, being present in the pupal brain, fat body, prothoracic gland and gut, as well as larval integumented epidermis, muscle and the wing imaginal discs. Most of the phosphoproteins stimulated by cAMP were localized in the microsomal fraction of tissue homogenates. The 31/32 kDa doublet phosphoproteins were further localized to a ribosome enriched microsomal fraction and have properties similar to those of mammalian ribosomal protein S6.

Serine protease activity has been extracted and partially purified from the alimentary tract of larval Helicoverpa armigera. The major activity was present in the midgut contents. Characterisation of this protease, using BApNA as substrate, gave a pH optimum of pH 9.5–10. K_m and V_max were 0.254 ± 0.032 mM and 0.351 ± 0.037 μmol pNA produced min⁻¹ mg protein⁻¹ respectively. Inhibition was effected by TLCK but not TPCK; this together with the failure to hydrolyse BTpNA or SUPHEPA, indicated trypsin-like but not chymotrypsin-like specificity. Comparison between the insect protease and bovine trysin revealed differences in inhibitor sensitivity; the insect protease being unaffected by OMTI, whilst showing greater inhibition by chymostatin and SBTI. The kinetics of the interactions between the insect protease activity and various plant-derived protease inhibitors were determined. Unlike bovine trypsin, the insect enzyme was not affected by calcium ions or the divalent chelating agent, EDTA. Partial purification by ion-exchange chromatography, followed by SDS-PAGE, showed that protease activity was largely associated with a polypeptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{⋍ 24,000}$.