Insect Biochemistry最新文献

Transcripts of three actin genes accumulate differentially during development of Bombyx mori, indicating distinct patterns of expression for each gene. Two of them are muscle actin encoding genes since one is expressed only in adult muscles which are formed during the late pupal period and the other one is expressed in all the larval as well as adult muscles tested. The third one codes for a cytoplasmic actin present particularly in embryos, larval silk glands and pupae. The structure of the 3′ end of each gene has been determined and indicates the presence of regulation in the choice of polyadenylation sites for the larval and adult muscle actin gene at different developmental stages. The pattern of accumulation of the Bombyx actin transcripts during development has been compared to that of Drosophila actin genes and indicates that three classes of actin genes can be distinguished in these two insect species: adult muscle actin genes, larval and adult muscle actin genes and cytoplasmic actin genes.

The induction of trypsin activity in the midgut of the mosquito, Aedes aegypti, was studied following meals of chicken blood, and several protein and peptide diets. Various concentrations of bovine serum albumin (BSA) in 0.15 M NaCl stimulated trypsin activity, in a similar fashion to the initial increase observed after a normal blood meal. Trypsin synthesis was also initiated when Ae. aegypti were fed on glutaraldehyde cross-linked BSA and on BSA fragments prepared by both pepsin and cyanogen bromide cleavage. Non-soluble proteins, in the form of glutaraldehyde-fixed erythrocyte ghosts, induced a delayed and reduced trypsin response, whilst small peptides from neutralized liver digests did not induce trypsin activity until 8–10 h after feeding. Metabolic inhibitors had varying effects on the post-feeding activity of trypsin stimulated by BSA feeding. Cycloheximide, a peptidyl transferase inhibitor prevented expression of all activity in vivo, whereas α-amanitin (RNA-polymerase inhibitor) did not affect trypsin activity in the first 10 h after feeding. At 20 μg/ml concentration in the diet, actinomycin D (RNA synthesis inhibitor) caused temporary superinduction followed by inhibition of trypsin activity, but at lower concentrations, the later phase of trypsin activity was inhibited. The results suggest that post-feeding induction of trypsin activity in Ae. aegypti is a two-phase process regulated at the midgut cellular level. The first phase of trypsin synthesis is stimulated by soluble proteins of variable molecular weights, and only involves translation of messenger RNA already available within the midgut cells. The second phase is stimulated by small peptides and requires complete synthesis of new mRNA from DNA.

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-(p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.

A major peak of juvenile hormone esterase (JHE) activity approaching 330 nmol JH III hydrolyzed/min/ml of hemolymph was observed during the last larval growth stage in Lymantria dispar. A smaller peak of JHE occurred 3–5 days after pupation. The gypsy moth JHE was purified from larval hemolymph using a classical approach. A specific activity of 766 units per mg of protein and a K_m of 3.6 × 10⁻⁷ M for racemic JH III and the (10R, 11S) enantiomer of JH II was determined for the purified enzyme. The 62 kDa esterase was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate (DFP), or by phenylmethylsulfonyl fluoride (PMSF). Two forms of JHE isolated by RP-HPLC were indistinguishable by HPLC tryptic peptide mapping and share an identical N-terminal amino acid sequence. Polyclonal antisera raised against gypsy moth enzyme cross-reacted with JHE from Trichoplusia ni but not with JHE from Manduca sexta. A weak cross-reactivity was observed with JHE from Heliothis virescens. Forty amino acid residues of the N-terminus were placed in sequence. The N-terminal sequence of JHE from L. dispar showed little homology to the sequence of JHE from H. virescens. The immunological and structural data support the conclusion that markedly different esterases, which catalyze the hydrolysis of juvenile hormone, are present in the hemolymph of different Lepidoptera.

The reproductive tissues of the primitive insect Thermobia domestica synthesize several eicosanoids when incubated with exogenous arachidonic acid. The enzymatic system was characterized with respect to kinetic parameters, Ca²⁺ requirement and pH- and cofactor-dependence. Five monohydroxylated eicosatetraenoic acids (HETEs) were identified by mass spectrometry procedures, demonstrating the existence of the lipoxygenase pathway. Quantitative studies were performed by high-pressure liquid chromatography. It is confirmed that the 8-lipoxygenase activity remains the major pathway in tissues from males and from inseminated females. In female tissues, the amounts of metabolite depend on the number of spermatophores contained in the seminal receptacles. These data are in agreement with the hypothesis of a transfer of the enzyme from male to female during mating. The mechanisms involved are discussed in comparison with those of other insect species in which the synthesis of prostaglandins has been reported.

The accumulation of transcripts of a cytoplasmic actin gene, the actin A3 gene, has been studied in relation to the silk production cycle in the two secretory regions of the silk glands of Bombyx mori. This accumulation was roughly similar in the two regions. The cellular amount of actin A3 mRNA decreased during the first period of the fourth moult, increased on and after the second period of this moult to reach a maximum at Day 5 of the fifth instar, then finally decreased. Actin biosynthesis almost completely ceased in the first period of the fourth moult and recovered in the second period of this moult. The accumulation of actin A3 mRNA parallels the development, in the silk gland cells, of the apical network of microfilaments which are implicated in silk secretion and which are disrupted at the beginning of the moult period and recorganized during the intermoult period. The accumulation of transcripts from actin A3 gene was compared to that of transcripts from silk protein genes and they were found to be asynchronous during the secretory cycle, indicating that mechanisms regulating expression of these two types of genes are different in the silk glands.

Purine interconversions leading to urate synthesis were studied over 60 min in isolated fat bodies from freshly collected Nasutitermes walkeri using ¹⁴C-hypoxanthine, ¹⁴C-inosine and ¹⁴C-guanosine. All were taken up, inosine the most efficiently at an initial rate of 0.06 ± 0.009 nmol/min/mg protein. The major purines, nucleosides and nucleotides were separated and examined for radioactivity. Based on uptake data, 1.3% of ¹⁴C-hypoxanthine, 0.3% of ¹⁴C-inosine and 37% of ¹⁴C-guanosine were converted to urate while 3% of the ¹⁴C-guanosine taken up was also salvaged to nucleotides. Feeding experiments with allopurinol showed that there was no significant production of urate via guanine, guanosine and adenosine. Incorporation data indicated the presence of the enzymes purine nucleoside phosphorylase, xanthine dehydrogenase, guanase and that neither inosine nor hypoxanthine could be salvaged.

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.

ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.

Epoxide hydrolase (EH) activity in Drosophila melanogaster cell fractions was characterized using juvenile hormone (JH III) and $\text{cis-}\text{stilbene}$ oxide (CSO) as substrates. A comparison of detergents indicated that 0.3% Lubrol PX was relatively effective for solubilizing EH activity from the 20,000 and 100,000 g pellets. The effect of inhibitors, pH, temperature, salt and organic solvents on EH activity depended on the substrate and cell fraction tested, which suggested there were multiple activities present. For initial purification, polyethylene glycol was useful for precipitating EH activity from the 100,000 g supernatant.

Follicle cells were isolated from their oocytes using 0.15% collagenase and low speed centrifugation. Incubation of the follicle cell pellet with [³H]2-deoxyecdysone yielded its 22-phosphate ester conjugate. Addition of ATP/Mg²⁺ or GTP/Mg²⁺ to follicle cell homogenate incubated with 2-deoxyecdysone increased the phosphotransferase activity by 4-fold for ATP and 2-fold for GTP. In the case of intact cells, only ATP was effective. The enzyme had a cytosolic subcellular localization and its $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for 2-deoxyecdysone was 3 μM. The phosphotransferase activity required the presence of both ATP and Mg²⁺, and had an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of 0.83 mM, with maximum activity being obtained in the presence of 10 mM Mg²⁺. The activity was strongly inhibited in the presence of Ca²⁺ with $\text{IC}{}_{50}\text{= 1}$ mM. The reaction rate was linear for 10 min and with increasing protein concentrations up to 1 mg/ml. Optimal pH was about 7.4 and the optimal temperature was 37°C. The phosphotransferase activity survived freezing at −20°C, but was totally abolished by heat at 60°C for 10 min. Investigation of the variation in activity of the phosphotransferase during ovarian development revealed a peak at the end of oogenesis in excellent agreement with the titre of ecdysteroid 22-phosphates found in the oocytes just before chorionation and egg-laying.