maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

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186 CHAPITRE 5. ADAPTATIONS RESPIRATOIRES DE S. MESATLANTICA Sodium and potassium - Sodium and potassium concentrations are determined using a flame photometer (Radiometer FLM3, Copenhagen, Denmark). Calcium and magnesium - calcium and magnesium concentrations were determined using the colorimetric kits Ca-kit 61041 and Mg-kit 61411 from Biomérieux, France. L-lactate and urate - L-lactate and urate concentrations were determined using the L-lactic acid Enzytec fluid kit from Scil Diagnostics GmbH, Germany and the AU PAP 150 kit from Biomérieux, France. Hemocyanin purification Native hemolymph was analyzed by size-exclusion chromatography (SEC) using a Superose- 6 10/300 GL column (Amersham Bioscience) at an elution rate of 0.5 ml/min with a crustacean physiological saline buffer (500 mM NaCl, 10 mM KCl, 30 mM MgSO4, 20 mM CaCl2, 50 mM Tris, pH 7.8, modified from Chausson et al. (2004)). Absorbance was recorded at 280 nm and 340 nm for protein and Hc detection, respectively. Complex proportions were determined using the area under each Hc elution peak. For purification, native hemolymph was injected and complexes were separated using the same method but at an elution rate of 0.25 ml/min. Dodecameric and hexameric fractions were collected and concentrated on centrifugal filters (10 kDa Microcon - Millipore). Multi-angle laser-light scattering (MALLS) Native mass of the Hc complexes was determined using multi-angle laser-light scattering. A MALLS device (Dawn EOS, Wyatt Technology, USA) was connected at the end of the SEC system previously described, along with a refractive index (RI) detector (Waters 2414). The dn/dc value taken for Hc is 0.190 ml/g, typical of non-glycosylated proteins. Masses are determined using the Astra 4.90.08 software (Wyatt Technology, USA) and employing the Zimm fit method. Normalization of the signals from the MALLS detectors was performed using BSA (Sigma). Electrospray ionization mass spectrometry (ESI-MS) Hemocyanin samples were analyzed by ESI-MS in non-covalent (supramolecular ESI-MS) and in denaturing conditions in order to determine the masses of the complexes and of their constituting subunits, respectively. Analyses were performed on 25 individuals, for each of which whole hemolymph was analyzed in non-covalent and denaturing conditions and purified dodecamers and hexamers were analyzed in denaturing conditions. All samples used for ESI-MS were desalted through concentration-dilution cycles using 10 kDa Microcon (Millipore) prior to analysis. Typically, 30 µl of
5.4. MANUSCRIT : RESPIRATORY ADAPTATIONS OF S. MESATLANTICA 187 native hemolymph diluted in 470 µl 10 mM ammonium acetate (AcNH 4 ), pH 6.8 or 250 µl of purified sample (protein concentration about 1-3 µg/µl) were first concentrated to almost dryness and then resuspended in 400 µl 10 mM AcNH 4 . At least 10 successive similar concentration-dilution steps were performed in order to ensure that the samples were correctly desalted prior to ESI-MS analysis. The samples were stored at 4°C in 10 mM AcNH 4 until analysis. The desalting was realized just prior to MS analysis and desalted samples for non-covalent analysis were not kept more than 2 days at 4°C because of dissociation occurring upon longer storage. Mass spectrometry experiments were performed on a MicroTOF instrument (Bruker Daltonics, Bremen, Germany) equipped with an ESI source. For non-covalent analysis, desalted samples were diluted in 10 mM AcNH 4 at a final concentration of approximately 1.5 µM (for 900 kDa dodecamers). The injection rate for the samples was 4 µl/min. The nebulization voltage was set to 5 kV, nebulization gas (N 2 ) pressure was 1 bar, drying gas flow was 4 l/min, source temperature was 200°C and the capillary exit voltage was set to 400 V. The calibration was made using 1 mg/ml CsI in water/isopropanol (50 :50 volume). The acquisition range was 500 to 20000 m/z. Spectra were smoothed using a Savitzky-Golay method and baseline subtracted. Complex masses were estimated using an in-built ruler (Bruker DataAnalysis v3.2 software). For denaturing analysis, desalted samples were diluted in a water/acetonitrile/formic acid mix (H 2 O/ACN/FA 50 :50 :1 volume) at a final concentration of approximately 4 µM (for 75 kDa subunits). The nebulization gas (N 2 ) pressure was 0.3 bar, drying gas flow was 3 l/min, and the capillary exit voltage was set to 160 V. Calibration was performed using myoglobin (Sigma-Aldrich). Mass spectra were analyzed using maximum entropy deconvolution (Bruker DataAnalysis v3.2 software). Determination of O 2 -binding properties of hemocyanin Oxygen equilibrium curves were recorded with a modified diffusion chamber (Sick et Gersonde, 1969) using the step by step method (Bridges et al., 1979, Lallier et Truchot, 1989)). A 3-5 µl sample was used for saturation measurements ; gas mixtures (N 2 and O 2 ) were prepared using mass flow controllers (MKS Instruments) connected to a multi gas controller unit (647B) and the absorbance of the sample was monitored at 340 nm. Samples were washed in the crustacean physiological saline buffer described above by at least 7 concentration-dilution steps using centrifugal filters (10 kDa Amicon, Millipore). For determination of the magnesium effect, samples were washed in an Mg 2+ -free saline buffer and mixed with physiological saline buffer containing various concentrations of Mg 2+ . The Mg 2+ experiments were simultaneously led on Carcinus maenas hemocyanin. For determination of the Bohr effect and of the L-lactate and urate effects, the Hc samples washed in the saline buffer were mixed with identical buffer containing various concentrations of L-lactate (Sigma) and urate
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THESE DE DOCTORAT DE L’UNIVERSITE
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i Résumé / Abstract Réponse adap
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iv SOMMAIRE 4.2 Objectifs de l’é
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2 CHAPITRE 1. INTRODUCTION Les éco
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4 CHAPITRE 1. INTRODUCTION 1.1 Mili
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6 CHAPITRE 1. INTRODUCTION Les prin
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8 CHAPITRE 1. INTRODUCTION TAB. 1.1
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10 CHAPITRE 1. INTRODUCTION et cycl
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FIG. 1.5 - Localisation des princip
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(a) FIG. 1.6 - Fonctionnement d’u
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16 CHAPITRE 1. INTRODUCTION TAB. 1.
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18 CHAPITRE 1. INTRODUCTION FIG. 1.
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28 CHAPITRE 1. INTRODUCTION TAB. 1.
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30 CHAPITRE 1. INTRODUCTION 700 600
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32 CHAPITRE 1. INTRODUCTION férent
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42 CHAPITRE 1. INTRODUCTION presque
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52 CHAPITRE 1. INTRODUCTION amélio
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104 CHAPITRE 3. STRUCTURE DE L’HC
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134 CHAPITRE 4. PLASTICITÉ PHÉNOT
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Bibliographie Adair, G. S. The hemo
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BIBLIOGRAPHIE 239 Catalina, M. I.,
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BIBLIOGRAPHIE 241 Farmer, C. S., J.
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BIBLIOGRAPHIE 243 Hourdez, S. Adapt
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BIBLIOGRAPHIE 245 Mangum, C. P. Sub
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BIBLIOGRAPHIE 247 Rainer, J., C. P.
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BIBLIOGRAPHIE 249 Svedberg, T. et I
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BIBLIOGRAPHIE 251 Vanin, S., E. Neg
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Liste des figures 1.1 Cycle des mar
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LISTE DES FIGURES 255 4.17 Profils
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Table des matières Résumé Sommai
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TABLE DES MATIÈRES 261 4.3.5 Spect
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TABLE DES MATIÈRES 263 Table des m
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