Insect Ceratitis Capitata Research Articles

D-fructose 1,6-biphosphate aldolase from the dipteran Ceratitis capitata: Purification, physicochemical and enzymic properties

d -Fructose 1,6-biphosphate aldolase from the insect Ceratitis capitata was purified 99-fold to apparent homogeneity with an overall yield of about 31%. The final preparation had a specific activity in the forward direction of 11 μmol of d -fructose 1,6-biphosphate/min/mg of protein at 25°C. The K m values for d -fructose 1,6-biphosphate, d -fructose 1-phosphate, dihydroxyacetone phosphate, and d -glyceraldehyde 3-phosphate were, respectively, 4.3, 1500, 65, and 57 μM. Enzyme stability versus ionic strength and the dependence of the reaction velocity on temperature are also reported. Phosphate and 2-mercaptoethanol have been shown to be good protectors of the enzyme against inactivation. Extrapolation to zero protein concentration yielded a value of s 20.w =8.0 in sedimentation velocity experiments. The Stokes radius of the enzyme was calculated from molecular sieve chromatography to be 46.2 A. These data yielded a M r =158.000. Molecular weight determinations by sodium dodecyl sulfate gel electrophoresis gave a subunit molecular weight of 39,000, indicating a tetrameric structure for the enzyme. The isoelectric point of the protein was determined by electrofocusing and found to be 6.6. The amino acid composition and the-sulfhydryl group content was determined. The extinction coefficient at 280 nm was calculated by dry weight measurements and amino acid composition to be ∈ 1 1 mg cm /ml=1.00. Digestion with carboxypeptidase A gave the C-terminal sequence: -Leu-Ala-Tyr. CD studies on the far and near uv were also carried out on native insect enzyme. All these data are discussed in relation to the properties of d -fructose 1,6-biphosphate aldolase from other sources.

Fatty acid synthetase complex from the insect Ceratitis capitata

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1- 14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5–6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2 × 10 5. The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants ( V and K m ) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42 ± 1-SH residues and one phosphopantetheine moiety per 5.2 × 10 5. Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0–18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.

L-glycerol-3-phosphate dehydrogenase from the insect Ceratitis capitata Purification, physicochemical and enzymic properties

Soluble l-glycerol-3-phosphate dehydrogenase ( sn-glycerol-3-phosphate: NAD + 2-oxidoreductase, EC 1.1.1.8) from the Mediterranean fruit fly Ceratitis capitata has been purified 130-fold with an overall yield of about 40%. The final preparation had a specific activity of about 200 μmol NADH/min/mg protein. The enzyme preparation has been shown to be homogeneous throughout disc gel electrophoresis, dodecyl sulphate gel electrophoresis, isoelectric focusing and ultracentrifugation. The K m values for dihydroxyacetone phosphate, NADH, l-glycerol-3-phosphate and NAD + were respectively 0.33, 0.018, 0.74 and 0.26 mM. l-glycerol3-phosphate dehydrogenase from the insect had a maximal activity around pH 6.6 for the oxidation of NADH and pH 10.0 for the reduction of NAD +. It was stable from pH 6.0 to pH 9.0 at 20°C for I h and remained active after incubating at 30°C for 30 min at pH 6.6. The enzyme was completely inactivated by incubating at 60°C for 5 min. Enzyme stability versus ionic strength as well as the dependence of the reaction velocity on temperature are also reported. The active enzyme was found to have a minimum molecular weight of approx. 63 000. Molecular weight determinations by sodium dodecyl sulphate gel electrophoresis gave subunit weights of 33 500. The isoelectric point of the protein was determined by electrofocusing and found to be 5.75 ± 0.05. The extinction coefficient at 278 nm was calculated by dry weight measurements to be E 1mg/ml 1 cm = 0.42 ± 0.1. Sedimentation velocity studies on ultracentrifuge indicated a dependence of the sedimentation coefficient on the enzyme concentration. The amino acid composition of the enzyme was determined. The protein has no free N-terminal residue and the digestion with carboxypeptidases gave the C-terminal sequence: -Ala-Gly-Ser. All these data are discussed in relation to the properties of the enzyme from other sources.

Biochemistry of development in insects. Triacyglycerol and phosphoglyceride biosynthesis by subcellular fractions.

1. A different biochemical behaviour has been previously shown during several stages of the development of the insect Ceratitis capitata; thus, the acyl transferase activity has a different participation in the pathways of synthesis of triacylglycerols and phosphoglycerides by either larval or pharate adult stages of development. The results reported here are concerned with the behavior of mitochondria and microsomes from larvae and pharate adults for the synthesis of triacylglycerols and phosphoglycerides. In all preparations of either mitochondrial or microsomal fractions the optimum protein amount, previously determined, has been used. 2. The highest incorporation of [14C]palmitate into triacylglycerols and phosphoglycerides was achieved by the microsomal fraction from the larval stage of development of the insect. However, mitochondrial fractions from pharate adults utilized higher levels of [14C]palmitate than the microsomal preparation. These differences were not correlated with the palmitoyl-CoA synthetase activity. 3. Glycerol 3-phosphate influenced the fatty acid incorporation in a different manner depending on the stage of development of the insect. Increasing concentrations of glycerol 3-phosphate stimulated the synthesis of triacylglycerols by microsomal and mitochondrial preparations. This effect was not exhibited by the subcellular preparations of pharate adults. 4. Double-label experiments using [14C]glycerol 3-phosphate and[3H]palmitate showed qualitative differences in the synthesis of triacylglycerols by mitochondrial preparations from either larvae of pharate adults. Incorporation of the labelled fatty acids to endogenous triglycerides was preferentially shown by the mitochondria larval preparations. The pathways of synthesis of triacylglycerols and phosphoglycerides by microsomal preparations showed only quantitative differences depending on the stage of development of the insect. 5. Acylation of 14C-labelled lyso-phosphatidylcholine and 14C-labelled lyso-phosphatidylethanolamine by [3H]olate was assayed in the presence of miochondrial and microsomal preparations from the different stages of development of the insect. Microsomal fractions showed an efficient acyl transferase activity mainly when lyso-phosphatidyl-ethanolamine was used as a substrate.

Cyclic AMP during development and ageing of the insectCeratitis capitata

Cyclic AMP was determined during the development of the diptera Ceratitis capitata. The concentration of the nucleotide reaches a peak at apolysis with a sharp decline in the pharate adult stage. A gradual increase takes place through the longevity of adult stage reaching a maximum plateau at the end of life.

Cyclic nucleotide cyclase variation during development of the insect Ceratitis capitata.

Abstract Guanylate and adenylate cyclase activities were estimated in homogenates of the insect Ceratitis capitata at various stages of development. Guanylate cyclase activity was notably higher than adenylate cyclase activity in agreement with both cyclic nucleotide ratio and cyclic nucleotide-dependent protein kinase ratio reported in arthropod tissues. Variations in both enzyme activities during development were coincident in the adult development, while in other biological stages, as the larval development and puparium formation, the most significant changes affected to the activity of guanylate cyclase.

Biochemistry of development in insects. Incorporation of fatty acids into different lipid classes.

1. To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time-course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged. 2. All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect. 3. Plots of triacylglycerol and free fatty acid radioactivites versus the stage of development defined a crossing-zone in coincidence with the larval-pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl-CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid. 4. [14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates. 5. Double-label experiments were carried out by incorporating either [3H]oleic acid or [3H]-palmitic acid and [14C]glycerol 3-phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.

Adenosine 3′,5′-monophosphate phosphodiesterase activity during development of the insect Ceratitis capitata

Summary Cyclic AMP phosphodiesterase activity was determined in homogenates of the insect Ceratitis capitata at several stages of development. These phosphodiesterase activities are interpreted on the bases of cyclic AMP concentrations and adenyl cyclase activities previously determined at the same stages of development. Variations of both enzyme activities are discussed in relation to the hormonal regulation of post-embryonic development in insects. The experimental results point to the possible existence of two sets of enzymes exhibiting different affinities for the naturally occurring cyclic nucleotides.

Primary structure of cytochrome c from the insect Ceratitis capitata

The complete amino acid sequence of cytochrome c from the Dipterous Ceratitis capitata (serie Acalypterae) has been determined by combining automatic and manual methods of sequence analysis. No overlaps between positions 79 and 80, 86 and 87, 91 and 92 as well as between 99 and 100 were obtained. The alignment of these peptides was done by homology with other sequences of cytochromes c from insects already described. Comparison with the sequences of cytochromes c of other Diptera studied so far shows three changes (positions 50, 60 and 61, according to vertebrate cytochrome c numeration) from the Acalypteran Drosophila melanogaster and five changes (positions 9, 36, 50, 60 and 61) from that of the Calypteran Haematobia irritans.

Automatic determination of the N-terminal sequence of cytochrome C from the insect Ceratitis capitata

Automatic determination of the N-terminal sequence of cytochrome C from the insect Ceratitis capitata

In vitro and in vivo [ 14C]acetate incorporation during development of insects

A comparative study on the in vitro and in vivo incorporation of [ 14C]acetate into different classes of lipids by the insect Ceratitis capitata has been carried out. The pattern of labelled lipids obtained in the in vitro experiments depends on both the time of incubation and the stage of development of the insect. Larval homogenates and pharate adult homogenates incorporate rapidly [1- 14C]acetate during the first 60 minutes, mainly into phospholipids. In the in vivo experiments triglycerides account for the highest percentages of incorporation in both stages of development, whereas free fatty acids and phospholipids exhibit lower levels of incorporation. Radioactivity of individual fatty acids present in the lipid classes was determined by gas-liquid radio-chromatography. The specific activity of fatty acids present as free fatty acids in triglycerides increased during the larval-pharate adult transition, whereas that of those present in phospholipids decreased. Relative abundance of monoenoic fatty acids synthesized de novo generally decreased from larvae to pharate adults. Triglycerides from both larvae and pharate adults showed the highest levels of unsaturation.

Changes of the positional distribution of fatty acids in phosphoglycerides during development of insects

The positional distribution of fatty acids in diacylcholine phosphoglycerides from the insect Ceratitis capitata during different developmental stages was investigated in relation to the previous data obtained from the ethanolamine phosphoglycerides. The total fatty acid pattern from eggs was similar to that of ethanolamine phosphoglycerides. Changes in total fatty acids during metamorphosis were clearly different from those in ethanolamine phosphoglycerides, but in both classes of phospholipids there was a net tendency to increase the degree of unsaturation. Fatty acid distribution both in the 1- and 2-positions of phosphatidylcholine showed some differences from the distributions observed in mammalian tissues ; thus, linoleic and linolenic acids occur in significant quantities at the 1-position of the phospholipid from larvae, pharate adults and adult insects. Position I of phosphatidylcholine exhibits a generally more pronounced tendency to join unsaturated fatty acids than that of phosphatidylethanolamine. These data suggested a different metabolic behaviour of acyl groups at the 1- and 2-positions in both classes of phospholipids.