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

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110 CHAPITRE 3. STRUCTURE DE L’HC DE C. MAENAS PAR ESI-MS Abstract The interaction of L-lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry in non-covalent conditions (supramolecular ESI- MS). Carcinus maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L-lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamerspecific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L-lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations.
3.2. MANUSCRIT : STRUCTURAL STUDY OF C. MAENAS HC BY ESI-MS 111 3.2.1 Introduction Hemocyanin, the blue respiratory pigment responsible for oxygen transport in decapod crustaceans, is a macromolecular complex comprised of 75 kDa subunits. These subunits can each reversibly bind one dioxygen molecule between the two copper ions of their active site and they associate into non-covalent complexes which can be hexamers (e.g. in isopods, krill, shrimps, prawns, spiny lobsters), dodecamers (e.g. in brachyurans, lobsters, crayfishes, hermit crabs) and in some species 24-mers (e.g. in thalassinid shrimps) (Markl, 1986, Markl et Decker, 1992, Terwilliger, 1998). Hemocyanin circulates as a freely dissolved protein in the hemolymph of the animals. Its functional properties can be modulated by various effectors such as H+, divalent cations and organic ions (Truchot, 1992, Bridges, 2001). Among them, the L stereoisomer of lactate is an important allosteric effector produced by the anaerobic metabolism of the animal, which can be driven by either environmental or metabolic hypoxia (Truchot, 1980, Truchot et Lallier, 1992). Divalent cations and pH have an effect both on the structure and on the properties of the complexes since most hemocyanins can be dissociated under alkaline pH and by dialysis against EDTA (Herskovits, 1988). Arthropod hemocyanins are useful models for studying allosteric regulation mechanisms. The existence of several types of allosteric effectors and the occurrence of huge hierarchical quaternary structures in some groups (e.g. 24-mers in arachnids, 48-mers in the horseshoe crab Limulus polyphemus (Markl et Decker, 1992)) allow for investigation of allosteric mechanisms with different molecular actors and using original models such as nested allostery (Decker, 1990, Menze et al., 2005). In most decapods, an increase in lactate concentration increases the oxygen affinity of the pigment (Truchot, 1992, Bridges, 2001, Truchot, 1980). However, some decapods exhibit low or no sensitivity to lactate (e.g. thalassinid mud-shrimps (Taylor et al., 2000), the slipper lobster Scyllarides latus (Sanna et al., 2004)), notably when the lifestyle becomes more terrestrial (Bridges, 2001, Morris et Bridges, 1994). For one terrestrial species, Gecarcoidea natalis, a reverse lactate effect has been observed (Adamczewska et Morris, 1998). The use of chemical analogs and the fact that D-lactate does not affect O2 affinity suggested that lactate binds stereospecifically to a specific site of hemocyanin (Johnson et al., 1984) and that the binding occurs at all four positions around the chiral carbon (Graham, 1985). For Callinectes sapidus hemocyanin, lactate titrations gave values of lactate dissociation constant of 1.8 mM for the oxy-state and 2.2 binding sites per hexamer, and ultrafiltration techniques
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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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20 CHAPITRE 1. INTRODUCTION Différ
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24 CHAPITRE 1. INTRODUCTION le mili
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26 CHAPITRE 1. INTRODUCTION Capacit
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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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34 CHAPITRE 1. INTRODUCTION (a) (b)
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40 CHAPITRE 1. INTRODUCTION 1 P 50
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42 CHAPITRE 1. INTRODUCTION presque
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44 CHAPITRE 1. INTRODUCTION affecte
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50 CHAPITRE 1. INTRODUCTION fure co
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52 CHAPITRE 1. INTRODUCTION amélio
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54 CHAPITRE 1. INTRODUCTION 1.5 Que
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56 CHAPITRE 2. ESI-MS ET MALLS APPL
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58 CHAPITRE 2. ESI-MS ET MALLS APPL
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160 CHAPITRE 4. PLASTICITÉ PHÉNOT
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Dénaturant Natif
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172 CHAPITRE 5. ADAPTATIONS RESPIRA
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216 CHAPITRE 6. CONCLUSION ET PERSP
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Annexe A Détermination des masses
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229 10000 h280 .M h) h280−h337 80
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Annexe B Détermination des distrib
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233 Modèle 2 : 2 sous-unités lég
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235 1 0.9 0.8 6 s-u aléatoires 2 s
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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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