Insect Biochemistry最新文献

A unique pattern of antioxidant enzymes exists in phytophagous insects for defense against endogenous and exogenous sources of toxic forms of oxygen, and data presented herein describe a profile of these enzymes in many tissues of larvae of the cabbage looper moth, Trichplusia ni. The specific activities of the antioxidant enzymes were high in tissues of high metabolic activities, i.e. Malpighian tubules, hindgut, muscles and gonads. A unique finding was the high constitutive activity of a superoxide dismutase (SOD) in hemocytes, probably consisting predominantly of the CuZn-SOD, which is analogous to the exclusive presence of this form of SOD in vertebrate erythrocytes and leukocytes. In all other tissues, the activity of Mn-SOD was higher than that of the CuZn-SOD which is converse to the pattern in vertebrate tissues. The glutathione peroxidase (GPOX) activity, present in all tissues and with highest levels in the gonads, did not seem to be the selenoprotein typical of the mammalian GPOX. $\text{Glutathione}\text{-S-}\text{transferase}$ (GST) activity paralleled that of its glutathione peroxidase activity (GSTPX). The high GSTPX activity suggests that GSTPX and not GPOX, forms a sequential team with glutathione reductase (GR) to reduce deleterious lipid hydroperoxides and to reduce the oxidized glutathione, GSSG, back to GSH. Catalase (CAT) which decomposes H₂O₂ has very high activity apparently correlated with the low GPOX activity. Finally, the integumental epithelium, and the gut (combined sections) possessed higher amounts of antioxidant enzymes than other tissues. Thus, a physiological relationship may occur between the antioxidant enzyme levels in tissues of T. ni with particularly high metabolic activity and associated endogenous oxidative stress. In addition, another physiological role of these enzymes may be to protect from exogenous oxidative stress exerted by dietary redox-active pro-oxidants in the gut, and to the potential of photodynamically mediated oxygen toxicity in peripheral organs such as the integument.

Using ethanol-chloroform fractionation in conjunction with standard column chromatography techniques catalase has been purified to electrophoretic homogeneity from mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni. The specific activity of purified catalase was 2.2 × 10⁵ units (IU = 1 μmol H₂O₂ decomposed mg protein⁻¹ min⁻¹). The purified enzyme's native molecular weight was in the 247,000–259,000 Da range and was tetrameric with an apparent molecular weight of 63,000 Da for each subunit. In addition, biochemical properties of the enzyme were studied with emphasis on substrate specificity, kinetics, and the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole (AT). The apparent K_m of the purified catalase for H₂O₂ was 54.2 mM and 50% of the maximal rate occurred at 16 mM H₂O₂. Purified catalase was ineffective in metabolizing organic hydroperoxides and, unlike other catalases, lacked peroxidase activity. Lastly, AT in the presence and absence of H₂O₂ was an effective inhibitor of catalase activity (I₅₀ = 100 mM) suggesting that a portion of the purified catalase was complexed with hydrogen peroxide in a compound 1 configuration.

The amount of glutathione S-transferase activity present in various stages of development and in various tissues of an insecticide susceptible strain of Helicoverpa zea was determined. GSH S-transferase activity toward CDNB (1-chloro-2,4-dinitrobenzene) was detected in the egg, and increased throughout the larval stages and was the highest in the 2 day old fifth instar larvae. GSH S-transferase activity toward DCNB (1,2-dichloro-4-nitrobenzene) could only be detected in the 2 day old fourth instar larvae, and the 2 day old fifth instar larvae. Enzyme activity using CDNB and DCNB as substrates was found in different tissues of the 2 day old fifth instar larvae, i.e. fat body, gut and cuticle. The fat body contained the highest enzyme activity for both substrates. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for CDNB conjugation in different stages of larval development and in the different tissues of the 2 day old fifth instar larvae, was approx. 0.24 mM. The enzyme responsible for CDNB-conjugation was purified 48 fold by GSH-agarose affinity chromatography. SDS gel electrophoresis and chromatofocusing resolved only one isozyme which is a homodimer, consisting of two equal size subunits having 23,900 mol. wt, with a pI value of 8.0.

Two cyclic nucleotide-dependent protein kinases have been purified and characterized from neural tissue of the honeybees Apis mellifera. The first kinase ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 180,000}$) has a tetrameric structure with two regulatory and two catalytic subunits that dissociate upon addition of cAMP. The regulatory subunits exist in phospho- and dephosphoform, which show electrophoretic mobilities in SDS-PAGE corresponding to $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 50,000}$ and $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 49,000}$, respectively. The values for half-maximal stimulation using histone IIA as substrate is 3.3 nM cAMP or 33 nM cGMP. The second cyclic nucleotide-dependent protein kinase, with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 80,000}$, is dimeric with a regulatory (phosphoform $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 36,000}$) and a catalytic subunit. Detection of this regulatory subunit in heads and brains homogenized in presence of SDS, TCA and various protease inhibitors supports strong evidence that the dimeric kinase is no artefact but exists in vivo. In contrast to the regulatory subunit of the tetrameric kinase, the regulatory subunit of the dimeric kinase is localized only in the soluble fraction.

The voltage-sensitive sodium channel is the site of action of two important classes of insecticides, DDT and pyrethroids. We recently used the polymerase chain reaction (PCR) to amplify sodium channel gene sequences in the house fly genome and showed the direct use of the amplification product as a conspecific hybridization probe. This report describes the use of this method to isolate sodium channel gene sequences from seven insect species (representing four orders) and an arachnid, thereby demonstrating its general utility for quickly and efficaciously isolating homologous sequences from distantly related species. DNA sequence analysis of the amplified products revealed that all but a few were homologous to the IS5-6 region of the para gene of Drosophila melanogaster, the region upon which the design of the target primers was based. Although unique nucleotide sequences were obtained for each species (with some species having more than one sequence variant), the inferred amino acid sequences of the 15 residue stretch between the amino acid target sequences were found to be completely conserved or to contain a single conservative replacement of serine with threonine. We suggest that this methodology now permits specific knowledge obtained from molecular genetic analysis of D. melanogaster to be applied straightforwardly to the characterization of many genes and the primary products of their expression in other insect specs.

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.

Ca²⁺-dependent proteinase activity was detected in the neural tissue of the honeybee Apis mellifera. DEAE-chromatography revealed two Ca²⁺-dependent proteinase activities, with elution positions corresponding to vertebrate calpain I and II. Both activities yielded apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 80,000}$, had a strict requirement for Ca²⁺ and a SH-reducing agent. PMSF and trypsin inhibitor did not affect the proteinase activities. Alkylating agents and mercaptide forming compounds as well as the calpain inhibitor calpastatin purified from bovine brain inhibited the activity. The natural inhibitor protein detected in the neural tissue of the honeybee showed properties equal to calpastatin from other sources. All observed properties are in accordance with those of the Ca²⁺-dependent proteolytic system calpain-calpastatin.

The transparent accessory reproductive gland of Rhodnius prolixus synthesizes and accumulates a variety of polypeptides. Ouchterlony immunodiffusion demonstrates that the hemolymph contains proteins which react with polyclonal antibodies against extracts of transparent accessory glands. Accessory glands and hemolymph contain a 170 kDa polypeptide with similar mobility on SDS-polyacrylamide gel electrophoresis. This polypeptide reacts with antibodies against extracts of accessory glands. Surgical removal of the accessory glands prevents the appearance of the 170 kDa polypeptide in the hemolymph. In vivo labeling of accessory gland proteins with a mixture of [¹⁴C]amino acids demonstrates that the newly synthesized TARG polypeptide appears in the hemolymph between days 2 and 3 after feeding. It is concluded that a specific polypeptide which is synthesized in the transparent accessory gland is exported to the hemolymph.

Larvae and pupae of Drosophila melanogaster are light sensitive. Light increases mortality and retards development from egg to adulthood. This effect of light is diet dependent. High yeast concentrations protect larvae and pupae against the injurious effect of light. At low yeast concentrations the larvae and pupae become extremely light sensitive. Addition of vitamins to a low yeast medium also gave protection against light. A first analysis revealed that vitamin H (biotin) is involved in the protection. This is shown by the effect of avidin, an inhibitor of biotin dependent enzymes, on the development time. In all these aspects we recorded significant differences between two strains, a control strain C and a strain P. The latter had been adapted to a palmitic acid supplemented medium. In addition to the effect of vitamins on the survival, amino acids and sucrose supplements protect the control strain. These supplements have no effect on the survival of the P strain at high light intensities. At low light intensities they improve the survival of both strains. Palmitic acid supplements affect the survival of the control strain at both light intensities approximately to the same extent. The P strain is affected by palmitic acid only under the low light conditions.