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A M Municio

Publications and source records attributed to A M Municio.

At least 73 records · Page 4Linked to original sources
PubMed1981

Conformational stability of fructose-1, 6-biphosphate aldolase from Ceratitis capitata.

Fructose 1, 6-biphosphate aldolase from Ceratitis capitata is a tetramer of identical subunits with 34% alpha-helix, 22% beta structure and 44% of aperiodic order. Increase of urea concentration up to 4.0 M results in non-cooperative reversible dissociation of the enzyme. Sodium dodecylsulphate 0.06% (w/v) dissociates the tetramer cooperatively with retention of the helical content. Thermal denaturation was a non-reversible cooperative process with a midpoint for the transition at 55 degrees. Cysteine residues are involved in this process and 2-mercaptoethanol preserves partially the enzyme activity. The acidic dissociation of the enzyme is a non-reversible process in contrast to the reversible basic dissociation. Increase of ionic strength results in a more ordered secondary structure for the monomer after acidic dissociation.

Animals↗
PubMed1979

Lipid requirements for the structure and function of the fatty acid synthetase complex from Ceratitis capitata.

Lipid content of purified fatty acid synthetase preparations from the Dipterous Ceratitis capitata correlated with the enzyme activity. Delipidation of the enzyme by extracting with a series of organic solvents rendered a protein without any residual activity and the treatment with phospholipase A2 for 30 min reduced the activity to 10%. Addition of lipid classes to either the native enzyme or the phospholipase-treated preparation enhanced the activity in a different manner, phosphatidylethanolamine being the most effective lipid. The role of the lipids in the lipoprotein structure of the complex was studied by circular dichroism spectra of the native enzyme and in the presence of a concentration range of urea, guanidinium chloride, sodium dodecyl sulfate, sodium cholate, and sodium chloride. 3 M urea and 1.5 M guanidinium chloride induced a conformational transition of the lipoprotein that lost its alpha-helical structure at higher concentrations of both reagents. Sodium dodecyl sulfate and sodium cholate had little effect on the alpha-helical structure, although both reagents induced the loss of enzyme activity. Cholate had essentially the same effect as phospholipids on the maintenance of the native structure but it was unable to support the enzyme activity.

Animals↗
PubMed1977

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 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(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.

Amino Acids↗
PubMed1977

L-glycerol-3-phosphate dehydrogenase from the insect Ceratitis capitata. Purfication, 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 mumol 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 Km values for dihydroxyacetone phosphate, NADH, L-glycerol-3-phosphate and NAD+ were respectively 0.33, 0.018, 0.74 and 0.26 mM. L-glycerol-3-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 degrees C for 1 h and remained active after incubating at 30 degrees C for 30 min at pH 6.6. The enzyme was completely inactivated by incubating at 60 degrees 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 E1cm 1mg/ml = 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.

Amino Acids↗
PubMed1977

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.

Animals↗
PubMed1977

Lysozyme from the insect Ceratitis capitata eggs.

1. Lysozyme from eggs of the Dipterous Ceratitis capitata (Wiedeman) has been purified by ion-exchange chromatography and gel filtration and its physicochemical properties have been investigated. This is the first insect lysozyme characterized so far and it exhibits some properties different to those described for other animal lysozymes. 2. Lysozyme from the insect eggs has a molecular weight of about 23200 and a sedimentation coefficient of 2.4 S. Molecular weight determination by sodium dedecylsulphate gel electrophoresis indicates that the molecule consists of a single polypeptide chain. 3. This lysozyme preparation shows notable stability at acidic pH values and lability at alkline pH values. It shows a single optimum pH at about 6.5.4. Chitinase/muramidase specific activity ratio is around 350 times higher for the insect lysozyme than for the hen egg-white enzyme. 5. The amino-acid composition shows the presence of one tryptophan residue per molecule of enzyme. This fact differentiates the lysozyme from insect eggs from other animal and plant lysozymes. From the amino acid composition, the absorption coefficient and the partial specific volume are calculated. 6. Glycine is the N-terminal residue.

Amino Acids↗
PubMed1976

Age differences in the positional distribution of phosphoglycerides and molecular species of choline phosphoglycerides during development of the chick embryo liver.

The total fatty acid composition and the distribution of acyl groups in the positions 1 and 2 of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine as well as in the molecular species of 1,2-diacyl-sn-glycero-3-phosphocholine from the chick embryo liver have been investigated at various stages of development. The fatty acid content of the main lipid classes from the yolks was also evaluated. The relative percentages of total saturated fatty acids in 1,2-diacyl-sn-glycero-3-phosphocholine remained constant during liver development; however, the levels of both main saturated acids, palmitic and stearic acids, were reciprocally counterbalanced as the age of the embryo proceeded. On the other hand, arachidonic acid decreased with time whereas linoleic acid and docosahexaenoic acid doubled their proportions during development. The relative amounts of total saturated fatty acids from liver 1,2-diacyl-sn-glycero-3-phosphoethanolamine were also constant during all stages of development; however, palmitic/stearic acid ratio did not change with time in this phosphoglyceride. Fatty acid composition of the yolk phosphoglycerides did not change during embryonic development. It should be noted that marked differences between fatty acid composition of choline and ethanolamine phosphoglycerides from the embryo liver and those of the yolk phosphoglycerides were demonstrated. The general patterns of the positional distribution of the acyl groups in choline phosphoglycerides differed from those of ethanolamine phosphoglycerides. Palmitic and stearic acids were only positioned at position 1 in 1,2-diacyl-sn-glycero-3-phosphoethanolamine whereas both fatty acids did not acylate exclusively the position 1 in 1,2-diacyl-sn-glycero-3-phosphocholine.

Animals↗
PubMed1975

Biochemistry of development in insects. Stereospecific incorporation of fatty acids into triacylglycerols.

1. Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time-course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn-positions of triacylglycerols has been followed. 2. Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn-1 and sn-3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn-3 position at a higher rate than it acylated the other sn-positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates. Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3). 3. Kinetics of incorporation of labelled fatty acids into the sn-positions points to a non-random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.

Animals↗
PubMed1975

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.

Animals↗
PubMed1975

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.

Amino Acid Sequence↗
PubMed1975

On methylating activity of L-(methyl-14C)-methionine in metabolism of phospholipids by insect Ceratitis capitata.

The methylating activity of L-(methyl-14C)-methionine in different stages of developmenent of the insect Ceratitis capitata was studied in a series of in vitro and in vivo experiments. Larval and pharate adult homogenates of the insect were used in the in vitro conditions, and the utilization of the methyl group of methionine for the synthesis of different classes of phospholipids was evaluated. Incorporation of radioactivity in lipids by pharate adult homogenates was significantly higher than that by larval homogenates. In both cases, phosphatidyl ethanolamine showed the highest levels of radioactivity incorporation. Free bases from total lipid hydrolysates were resolved and identified by paper chromatography, and the labeling was investigated by radioactivity scanning of paper chromatograms. Significant differences were observed in the activity of both stages of development of the insect. Larval and pharate adult homogenates incorporated mainly the labeled methyl groups into ethanolamine. Monmethyl ethanolamine was the only methyl derivative that appeared in the hydrolysates of lipids synthesized by larval homogenates, whereas mono-,di-and trimethyl ethanolamine clearly were detected in those synthesized by pharate adult homogeneates. Administration of L-(methyl 14C)-methionine to larvae confirmed the existence of methylation reactions in the metabolic activity of the insect.

Animals↗