7 results on '"Puiroux, J."'
Search Results
2. Identification and characterization of two dipeptidyl-peptidase III isoforms in Drosophila melanogaster.
- Author
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Mazzocco C, Gillibert-Duplantier J, Neaud V, Fukasawa KM, Claverol S, Bonneu M, and Puiroux J
- Subjects
- Animals, Base Sequence, Central Nervous System enzymology, DNA genetics, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases chemistry, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases genetics, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Immunohistochemistry, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes isolation & purification, Isoenzymes metabolism, Kinetics, Mass Spectrometry, Molecular Weight, Solubility, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases isolation & purification, Drosophila melanogaster enzymology
- Abstract
Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti-(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming approximately 60% of both annotated D. melanogaster DPP III isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at approximately 2.6 and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin (Km approximately 4 microm) at the second N-terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts. more...
- Published
- 2006
- Full Text
- View/download PDF
Catalog
3. Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster.
- Author
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Mazzocco C, Fukasawa KM, Auguste P, and Puiroux J
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Cell Membrane metabolism, Cloning, Molecular, Cytosol metabolism, DNA, Complementary genetics, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases antagonists & inhibitors, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases genetics, Drosophila melanogaster genetics, Enzyme Inhibitors pharmacology, Genetic Vectors, Glycosylation, Hydrolysis, Immunohistochemistry methods, Molecular Sequence Data, Oligopeptides metabolism, Oligopeptides pharmacology, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Transfection, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Drosophila melanogaster enzymology, Neuropeptides
- Abstract
A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (Km approximately 4 micro m). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 +/- 0.15 micro m). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation. more...
- Published
- 2003
- Full Text
- View/download PDF
4. Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin.
- Author
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Mazzocco C, Fukasawa KM, Raymond AA, and Puiroux J
- Subjects
- Amino Acid Sequence, Animals, Blotting, Western, Cockroaches cytology, Cockroaches genetics, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases chemistry, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases genetics, Drosophila melanogaster enzymology, Drosophila melanogaster genetics, Expressed Sequence Tags, Membrane Proteins chemistry, Membrane Proteins genetics, Membrane Proteins isolation & purification, Membrane Proteins metabolism, Molecular Sequence Data, Protein Binding, Rats, Sequence Analysis, Protein, Cockroaches enzymology, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases isolation & purification, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Neuropeptides, Oligopeptides metabolism
- Abstract
Two proctolin-binding proteins solubilized from 1600 cockroach hindgut membranes were purified 1000-fold using five chromatography steps. Twenty-five micrograms of protein were recovered from the final size-exclusion chromatography as a single peak eluting at 74 kDa, whereas two major bands at 80 and 76 kDa were identified after silver staining of electrophoresis gels. The fragments, sequenced by tandem mass spectrometry and the Edman method, revealed a high homology with rat liver dipeptidyl aminopeptidase (DPP) III and a significant homology between the cockroach-purified proteins. From analysis of the Drosophila genome sequence database, it was possible to identify a putative DPP sharing high homology with the sequences obtained from the cockroach purified proteins and with the rat DPP III. Anti-(rat liver DPP III) Ig reacted specifically with both cockroach-purified proteins in Western blot analysis. The purified proteins removed the N-terminal dipeptide from the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) with a Km value of 3.8 +/- 1.1 microM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC50 = 0.68 microM). These results provide strong evidence that the cockroach-purified proteins represent an insect membrane DPP, presumably present in Drosophila, and that it is closely related to vertebrate DPP III. more...
- Published
- 2001
- Full Text
- View/download PDF
5. Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer.
- Author
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Mazzocco C and Puiroux J
- Subjects
- Animals, Binding Sites, Detergents, Electrophoresis, Polyacrylamide Gel, Insect Proteins chemistry, Membrane Proteins chemistry, Membrane Proteins isolation & purification, Neuropeptides chemistry, Protein Binding, Solubility, Cockroaches chemistry, Insect Proteins isolation & purification, Oligopeptides chemistry
- Abstract
A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography. more...
- Published
- 2000
- Full Text
- View/download PDF
6. Pharmacological studies of proctolin receptors on foregut and hindgut of Blaberus craniifer.
- Author
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Mazzocco-Manneval C, Kuczer M, Konopinska D, Fournier B, Loughton BG, and Puiroux J
- Subjects
- Adenylyl Cyclases metabolism, Animals, Cell Membrane chemistry, Cell Membrane drug effects, Cell Membrane enzymology, Cockroaches, Cyclic AMP metabolism, Dose-Response Relationship, Drug, Hydrolysis drug effects, Phosphatidylinositols metabolism, Protein Binding drug effects, Receptors, Neuropeptide classification, Receptors, Neuropeptide physiology, Second Messenger Systems drug effects, Stomach cytology, Stomach drug effects, Neuropeptides, Neurotransmitter Agents pharmacology, Oligopeptides pharmacology, Receptors, Neuropeptide metabolism, Stomach chemistry
- Abstract
Proctolin (Arg-Tyr-Leu-Pro-Thr) and proctolin analogs modified at position 1, 2, or 5 caused dose dependent contractions of Blaberus fore- and hindgut. The varying contractile effects between both tissues revealed the possible presence of receptor subtypes as identified by [GABA1]-proctolin. A single population of binding sites (Kd approximately 100 nM) was deduced from Scatchard analysis. In addition, nanomolar concentrations of proctolin induced a dose-dependent hydrolysis of phosphoinositides (PIns) augmented by GTPgammaS (1 microM) on foregut membranes but no accumulation of cAMP. Proctolin induced contractions are likely mediated via a phospholipase C linked to a heptahelical receptor bound to heterotrimeric G-proteins. more...
- Published
- 1998
- Full Text
- View/download PDF
7. The effect of proctolin analogues and other peptides on locust oviduct muscle contractions.
- Author
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Puiroux J, Pedelaborde A, and Loughton BG
- Subjects
- Amino Acid Sequence, Animals, Biological Assay, Female, In Vitro Techniques, Molecular Sequence Data, Muscle Contraction drug effects, Oviducts drug effects, Structure-Activity Relationship, Grasshoppers drug effects, Neuropeptides, Neurotransmitter Agents pharmacology, Oligopeptides pharmacology
- Abstract
The locust oviduct bioassay system was used to assess the ability of a variety of peptides to induce oviductal contractions. Proctolin analogues were three orders of magnitude less potent than proctolin. Proctolin supra-analogue and Arg-Tyr-Leu-Ala-Thr demonstrated high activity. Perhaps the most significant finding was the discrepancy between the high binding capacity of the proctolin analogue Arg-Tyr-Ser-Pro-Thr and its relatively low myotropic activity. This observation argues for a crucial role for the leucine residue in activating the proctolin receptor. Several other myotropic peptides were tested for their effect on oviduct contractions. FMRFamide caused contractions at doses several orders of magnitude higher than proctolin. The FLRFamide leucomyosuppression inhibited proctolin-induced contractions. In addition, myomodulin and catch relaxing peptide caused oviductal contractions at low concentrations. The enkephalins had no effect when applied alone but potentiated proctolin-induced oviduct contractions. The mechanism of the potentiation is not known. The data argue for the presence of several binding sites on the oviduct membrane. more...
- Published
- 1993
- Full Text
- View/download PDF
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