tailieunhanh - Báo cáo khoa học: Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex – purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga,Chlamydomonas rein-hardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC ), glyceraldehyde 3-phosphate dehydrogenase (EC ) and aldolase. | ễFEBS Journal Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase glyceraldehyde 3-phosphate dehydrogenase CP12 complex - purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii Jenny Erales1 Luisana Avilan2 Sandrine Lebreton3 and Brigitte Gontero1 1 Laboratoire d Enzymologie de Complexes Supramoleculaires UPR 90 36 BIP-CNRS Marseille France 2 Departamento de Biologia Facultad de Ciencias Universidad de Los Andes Merida Venezuela 3 Institut Jacques Monod UMR 75 92 CNRS-Universites Paris VI et VII France Keywords CP12 FBP aldolase GAPDH IUP PRK Correspondence B. Gontero Laboratoire d Enzymologie de Complexes Supramoleculaires UPR 90 36 BIP-CNRS 31 Chemin J. Aiguier. 13402 Marseille Cedex 20 France Fax 33 4 91 16 46 89 Tel 33 4 91 16 45 49 E-mail bmeunier@ Received 7 November 2007 revised 14 December 2007 accepted 9 January 2008 doi Possible binding proteins of CP12 in a green alga Chlamydomonas rein-hardtii were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus we found an association between CP12 and phosphoribulokinase EC glyceraldehyde 3-phosphate dehydrogenase EC and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1 6-bisphosphate EC aldolase was measured by surface plasmon resonance and is equal to pM and thus corroborated an interaction between CP12 and aldolase. However the association is even stronger between aldolase and the phospho-ribulokinase glyceraldehyde 3-phosphate dehydrogenase CP12 complex and the dissociation constant between them is equal to 55 5 nM. Moreover owing to the fact that aldolase has been poorly studied in C. reinhardtii we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1 .