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- Title
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.
- Authors
Erales, Jenny; Avilan, Luisana; Lebreton, Sandrine; Gontero, Brigitte
- Abstract
Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, 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 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 ± 0.05 μm and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/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 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 ± 1 s−1 and 4 ± 0.1 s−1, respectively. The Km value for fructose 1,6-bisphosphate was equal to 0.16 ± 0.02 mm and 0.046 ± 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 ± 31 s−1·mm−1 with fructose 1,6-bisphosphate and 87 ± 9 s−1·mm−1 with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 ± 23 s−1·mm−1, whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.
- Subjects
CHLAMYDOMONAS reinhardtii; GREEN algae; CARRIER proteins; SURFACE plasmon resonance; BIOSENSORS
- Publication
FEBS Journal, 2008, Vol 275, Issue 6, p1248
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2008.06284.x